High resolution imaging apparatus and method

ABSTRACT

The present invention relates to the high resolution imaging of samples using imaging mass spectrometry (IMS) and to the imaging of biological samples by imaging mass cytometry (IMCTM) in which labelling atoms are detected by IMS. LA-ICP-MS (a form of IMS in which the sample is ablated by a laser, the ablated material is then ionised in an inductively coupled plasma before the ions are detected by mass spectrometry) has been used for analysis of various substances, such as mineral analysis of geological samples, analysis of archaeological samples, and imaging of biological substances. However, traditional LA-ICP-MS systems and methods may not provide high resolution. Described herein are methods and systems for high resolution IMS and IMC.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. ProvisionalApplication No. 62/686,341 filed Jun. 18, 2018 and entitled “HIGHRESOLUTION IMAGING APPARATUS AND METHOD,” and U.S. ProvisionalApplication No. 62/792,759 filed Jan. 15, 2019 and entitled “HIGHRESOLUTION IMAGING APPARATUS AND METHOD,” both of which are herebyincorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to the high resolution imaging of samplesusing imaging mass spectrometry (IMS) and the imaging of biologicalsamples by imaging mass cytometry (IMC™).

BACKGROUND

LA_ICP-MS (a form of IMS in which the sample is ablated by a laser, theablated material is then ionised in an inductively coupled plasma beforethe ions are detected by mass spectrometry) has been used for analysisof various substances, such as mineral analysis of geological samples,analysis of archaeological samples, and imaging of biologicalsubstances.

Imaging of biological samples by IMC has previously been reported forimaging at a cellular resolution. Detailed imaging at a sub-cellularresolution has also recently been reported.

Despite these recent advances, there is a need for still furtherapparatus and techniques that enhance the resolution of IMC to submicrometer scales. The two main challenges in enhancing the resolutionof IMC to sub micrometer scales are:

-   -   1) confining the sampling spot area to a size of around 200 nm        or less in size; and    -   2) ensuring that the amount of analyte in the ablated material        produces a sufficient signal-to-noise ratio.

It is an object of the invention to provide further and improvedapparatus and techniques which overcome these two challenges in order toprovide for high resolution imaging of samples.

SUMMARY OF THE INVENTION

In general terms, the analyser apparatus disclosed herein comprises twobroadly characterised systems for performing imaging elemental massspectrometry.

The first is a sampling and ionisation system. This system contains asample chamber, which is the component in which the sample is placedwhen it is subjected to analysis. The sample chamber comprises a stage,which holds the sample (typically the sample is on a sample carrier,such as a microscope slide, e.g. a tissue section, a monolayer of cellsor individual cells, such as where a cell suspension has been droppedonto the microscope slide, and the slide is placed on the stage). Thesampling and ionisation system act to remove material from the sample inthe sample chamber (the removed material being called sample materialherein) which is converted into ions, either as part of the process thatcauses the removal of the material from the sample or via a separateionisation system downstream of the sampling system. To generateelemental ions, hard ionisation techniques are used.

The ionised material is then analysed by the second system which is thedetector system. The detector system can take different forms dependingupon the particular characteristic of the ionised sample material beingdetermined, for example a mass detector in mass spectrometry-basedanalyser apparatus.

The present inventions provide improvements over current IMS and IMCapparatus and methods by the application of various apparatus andtechniques which exploit advantages in the analysis of tissue sectionsof a thickness less than 100 nm. When such ultrathin sections are used,the opportunity for further analytical techniques is increased. Theinventors have discovered it is possible to use lenses which maintain aparticularly high refractive index around the ablation spot (e.g.immersion lenses), which focus the laser used for laser ablation down toa particularly small ablation spot. The inventors have determined thatthat when ultrathin sections are analysed, such lenses can be used tofocus laser radiation without causing damage to areas of the sampleoutside the area targeted for ablation. This is because the highrefractive index of the lens material ensures that the laser is focusedinto a tight focal spot all the way through the lens down to theablative spot. This tight focal spot is thus smaller than would bepossible without the high refractive index material and because the sizeof the focal spot determines lateral resolution, the present inventionthus provides apparatus and techniques which provide for improvedlateral resolution. Furthermore, the focus of the lens is not merely intwo dimensions. It is a three dimensional volume of concentratedradiation. Accordingly, the diameter, but also the depth of the ablativespot (i.e. the focal point of the radiation) is of concern. If the focallength is set such that the focus of the radiation is in the substraterather than at its surface, this can lead to unpredictable breakdown andfragmentation of the sample. This problem is caused in particular by thefact the inventors' finding that to use an immersion lens mosteffectively, the sample is best ablated in a manner that directs thelaser radiation through the sample carrier.

Likewise, the inventors have discovered that when thin sections areused, it is possible to perform electron microscopy on a sample alsoanalysed by IMS or IMC, for example, prior to analysing the sample byIMC. Accordingly, high resolution structural images can be obtained byelectron microscopy, for example transmission electron microscopy, andthen this high resolution image used to refine, the resolution of imagedata obtained by IMS or IMC to a resolution beyond that achievable withablation using laser radiation (due to the much shorter wavelength ofelectrons compared to photons). Alternatively or in addition, theelectron microscopy image may be related to the IMC image of the sameregion of a sample, for example by relating the localization of proteintargets imaged by IMC with subcellular (e.g., nano-scale) structuresidentified by electron microscopy. As described in more detail below,the electron microscopy imaging may be performed “off line” in aseparate apparatus, or the components of the electron microscope may beincorporated into the IMS or IMC apparatus.

Moreover, the inventors have developed further techniques for the highresolution imaging based on a combination of charged particles (e.g. ionor electron) bombardment of a sample coupled with laser ionisation ofthe sample material, either subsequent to the sputtering of samplematerial from the sample or at the time of sampling. As chargedparticles can be focussed more tightly while still achieving removal ofmaterial from the sample, again greater resolution can be achieved bythis means compared to traditional laser ablation based techniques. Ionoptics may be used to direct charged particles toward a sample, and/ordirect ionized sample to a mass spectrometer (e.g., a TOF or magneticsector detector). Ion optics may be similar to those used in electron orion microscopes. Ion optics may include ion scanning optics (e.g., forscanning charged particles across a sample), ion focusing optics (e.g.,for determining an spot size or focusing sample ions toward a detector),and ion accelerating optics (to determine energy of charged particlesimpinging the sample, or for accelerating ions toward a detector). Ionoptics may comprise one or more charged surfaces (e.g., plates) ofappropriate shape, charge and/or orientation.

Thus, in operation of one apparatus according to the invention, thesample is taken into the apparatus, is sampled to generate ionisedmaterial using a laser system comprising optics in which laser radiationis directed onto the sample though an immersion lens (sampling maygenerate vaporous/particular material, which is subsequently ionised bythe ionisation system), and the ions of the sample material are passedinto the detector system.

Thus, in operation of another apparatus according to the invention, thesample is taken into the apparatus, is imaged using by electronmicroscopy (e.g. transmission electron microscopy), and then is sampledto generate ionised material using a sampling and ionisation system(sampling may generate ions, or may generate non-chargedvaporous/particular material (or the non-charged vaporous/particularmaterial may be formed by charges neutralising in any ions formed onsampling), which in the latter case is subsequently ionised by aseparate ionisation system), and the ions of the sample material arepassed into the detector system.

Finally, in operation of a further apparatus of the invention, thesample is taken into the apparatus, is sampled to generate materialusing a sputtering based sampling system (a charged particle-basedsampling system), where the sample material is ionised by a laserpost-ionisation system, either subsequent to the sputtering of samplematerial from the sample or at the time of sampling, and the ions of thesample material are passed into the detector system.

Although the detector system of the apparatus of the invention candetect many ions, most of these will be ions of the atoms that naturallymake up the sample. In some cases, for example when analysing biologicalsamples, the native element composition of the sample may not besuitably informative. This is because, typically, all proteins andnucleic acids are comprised of the same main constituent atoms, and sowhile it is possible to tell regions which contain protein/nucleic acidfrom those that do not contain such proteinaceous or nucleic acidmaterial, it is not possible to differentiate a particular protein fromall other proteins. However, by labelling the sample with atoms notpresent in the material being analysed under normal conditions, or atleast not present in significant amounts (for example certain transitionmetal atoms, such as rare earth metals; see section on labelling belowfor further detail), specific characteristics of the sample can bedetermined. In common with IHC and FISH, the detectable labels can beattached to specific targets on or in the sample (such as fixed cells ora tissue sample on a slide), inter alia through the use of SBPs such asantibodies, nucleic acids or lectins etc. targeting molecules on or inthe sample. In order to detect the ionised label, the detector system isused, as it would be to detect ions from atoms naturally present in thesample. By linking the detected signals to the known positions of thesampling of the sample which gave rise to those signals it is possibleto generate an image of the atoms present at each position, both thenative elemental composition and any labelling atoms. In aspects wherenative elemental composition of the sample is depleted prior todetection, the image may only be of labelling atoms. The techniqueallows the analysis of many labels in parallel (also termedmultiplexing), which is a great advantage in the analysis of biologicalsamples, now with increased speed due to the application of a laserscanning system in the apparatus and methods disclosed herein.

Thus the invention provides an apparatus for analysing a biologicalsample comprising:

-   -   a sample stage;    -   a laser source; and    -   focusing optics comprising an objective lens, the focusing        optics adapted to direct a beam of radiation from the laser        source towards a location on the sample stage; and wherein    -   the apparatus further comprises an immersion medium positioned        between the objective lens and the sample stage.

The invention also provides a method of preparing a biological samplefor analysis comprising: staining the sample with a contrast agent forelectron microscopy; labelling the sample with labelling atoms.

The invention also provides a method of analysing a biological samplecomprising: imaging a sample by electron microscopy;

directing a beam of radiation emitted by the laser source towards alocation on the sample to produce an ablated plume of sample material;

ionising the ablated plume of sample material; and

detecting the sample ions from the sample material.

The invention also provides an apparatus for analysing a biologicalsample comprising: a sample stage;

a source of charged particles and a charged particle column for passinga beam of charged particles to a location on the sample stage; and

a first laser source and first focusing optics configured to direct alaser beam emitted by the first laser source towards the sample stage.

The invention also provides a method of analysing a biological samplecomprising: passing a beam of charged particles towards a location onthe sample to sputter material from the sample; illuminating thesputtered sample material with a pulse of laser radiation to produce aplume of material comprising sample ions; and detecting said sample ionsby mass spectrometry.

The invention also provides a method of analysing a biological samplecomprising: passing a beam of charged particles towards a location onthe sample produces a sample ignition state on the sample; illuminatingthe sample with a pulse of laser radiation to produce a plume of samplematerial comprising sample ions from the location; and detecting saidsample ions by mass spectrometry.

DESCRIPTION OF THE ACCOMPANYING FIGURES

FIG. 1 is a schematic diagram of the optics of a prior apparatus set up.

FIG. 2 is a schematic diagram of the optics of another prior apparatusset up.

FIG. 3 is a schematic diagram of the optics arrangement of an exemplaryembodiment of the invention.

FIG. 4 is a schematic diagram of the optics arrangement of a furtherexemplary embodiment of the invention.

FIG. 5a is a schematic diagram of the optics arrangement of anotherexemplary embodiment of the invention, illustrating the path of thelaser beam through a hemispherical solid immersion lens.

FIG. 5b is a schematic diagram of the optics arrangement of anotherexemplary embodiment of the invention, illustrating the path of thelaser beam through a Weierstrass solid immersion lens.

FIG. 6 is a schematic diagram of the optics arrangement of an apparatusfor analysing a biological sample using laser post-ionisation.

FIG. 7 is a schematic diagram of the optics arrangement of an apparatusfor analysing a biological sample using laser pumping of the sample.

FIG. 8 is a schematic diagram of the optics arrangement of an apparatusfor analysing a biological sample using laser pumping of the sample,through the sample carrier.

FIG. 9 is a schematic diagram of the optics arrangement of an apparatusfor analysing a biological sample using \ laser post-ionisation of thesample, through the sample carrier.

FIG. 10 is a schematic diagram of the optics arrangement of an apparatusfor analysing a biological sample comprising at least two laser sources,one for laser post-ionisation and one for laser pumping of the sample.

FIG. 11 shows an image showing the detection of various osmium isotopesin a sample prepared according to an exemplary method of the invention.

FIG. 12 shows an image showing the detection of various osmium isotopesin a sample prepared according to the exemplary method of the inventionas used for FIG. 11. FIG. 12 shows an area of 2100×1087 micrometres at astep size of 250 nm.

FIG. 13 is a schematic diagram of a two-pulse sampling and ionizationsystem.

FIG. 14 is a schematic diagram of a two-pulse sampling and ionizationsystem in which the first and second pulse are focused onto the specimenfrom the same side.

FIG. 15 is a schematic diagram of a two-pulse sampling and ionizationsystem in which the sample is resting on a material that is at leastsemi-transparent to the first and second pulse.

FIG. 16 is a schematic diagram of a two-pulse sampling and ionizationsystem in which the objective has an opening to allow passage of ionsgenerated after ablation.

FIG. 17 is a schematic diagram of a three-pulse sampling and ionizationsystem.

DETAILED DESCRIPTION OF THE INVENTION

Thus various types of analyser apparatus can be used in practising thedisclosure, a number of which are discussed in detail below forinstance, apparatus comprising immersion lenses, apparatus comprising anelectron microscope (or components thereof), and apparatus forperforming secondary neutral mass spectrometry.

Analyser Apparatus Based on Mass-Detection

1. Sampling and Ionisation Systems

a. Laser Ablation Sampling and Ionising System

A laser ablation based analyser typically comprises three components.The first is a laser ablation sampling system for the generation ofplumes of vaporous and particulate material from the sample foranalysis. Before the atoms in the plumes of ablated sample material(including any detectable labelling atoms as discussed below) can bedetected by the detector system—a mass spectrometer component (MScomponent; the third component), the sample must be ionised (andatomised). Accordingly, the apparatus comprises a second component whichis an ionisation system that ionises the atoms to form elemental ions toenable their detection by the MS component based on mass/charge ratio(some ionisation of the sample material may occur at the point ofablation, but space charge effects result in the almost immediateneutralisation of the charges). The laser ablation sampling system isconnected to the ionisation system by a transfer conduit.

Laser Ablation Sampling System

In brief summary, the components of a laser ablation sampling systeminclude a laser source that emits a beam of laser radiation that isdirected upon a sample. The sample is positioned on a stage within achamber in the laser ablation sampling system (the sample chamber). Thestage is usually a translation stage, so that the sample can be movedrelative to the beam of laser radiation, whereby different locations onthe sample can be sampled for analysis (e.g. locations more remote fromone another than can be ablated as a result of the relative movement inthe laser beam (the term laser beam can be used interchangeably with theterm laser radiation herein) can be induced by laser scanning systemdescribed herein). As discussed below in more detail, gas is flowedthrough the sample chamber, and the flow of gas carries away the plumesof aerosolised material generated when the laser source ablates thesample, for analysis and construction of an image of the sample based onits elemental composition (including labelling atoms such as labellingatoms from elemental tags). As explained further below, in analternative mode of action, the laser system of the laser ablationsampling system can also be used to desorb material from the sample.

For biological samples (cells, tissues sections etc.) in particular, thesample is often heterogeneous (although heterogeneous samples are knownin other fields of application of the disclosure, i.e. samples of anon-biological nature). A heterogeneous sample is a sample containingregions composed of different materials, and so some regions of thesample can ablate at lower threshold fluence at a given wavelength thanthe others. The factors that affect ablation thresholds are theabsorbance coefficient of the material and mechanical strength ofmaterial. For biological tissues, the absorbance coefficient will have adominant effect as it can vary with the laser radiation wavelength byseveral orders of magnitude. For instance, in a biological sample, whenutilising nanosecond laser pulses a region that contains proteinaceousmaterial will absorb more readily in the 200-230 nm wavelength range,while a region containing predominantly DNA will absorb more readily inthe 260-280 nm wavelength range.

It is possible to conduct laser ablation at a fluence near the ablationthreshold of the sample material. Ablating in this manner often improvesaerosol formation which in turn can help improve the quality of the datafollowing analysis. Often to obtain the smallest crater, to maximise theresolution of the resulting image, a Gaussian beam is employed. A crosssection across a Gaussian beam records an energy density profile thathas a Gaussian distribution. In that case, the fluence of the beamchanges with the distance from the centre. As a result, the diameter ofthe ablation spot size is a function of two parameters: (i) the Gaussianbeam waist (1/e²), and (ii) the ratio between the fluence applied andthe threshold fluence.

Thus, in order to ensure consistent removal of a reproducible quantityof material with each ablative laser pulse, and thus maximise thequality of the imaging data, it is useful to maintain a consistentablation diameter which in turn means adjusting the ratio of the energysupplied by the laser pulse to the target to the ablation thresholdenergy of the material being ablated. This requirement represents aproblem when ablating a heterogeneous sample where the thresholdablation energy varies across the sample, such as a biological tissuewhere the ratio of DNA and protein material varies, or in a geologicalsample, where it varies with the particular composition of the mineralin the region of the sample. To address this, more than one wavelengthof laser radiation can be focused onto the same ablation location on asample, to more effectively ablate the sample based on the compositionof the sample at that location.

Laser System of the Laser Ablation Sampling System

The laser system can be set up to produce single or multiple (i.e. twoor more) wavelengths of laser radiation. Typically, the wavelengths oflaser radiation discussed refer to the wavelength which has the highestintensity (the “peak” wavelength). If the system produces differentwavelengths, they can be used for different purposes, for example, fortargeting different materials in a sample (by targeting here is meantthat the wavelength chosen is one which is absorbed well by a material).

Where multiple wavelengths are used, at least two of the two or morewavelengths of the laser radiation can be discrete wavelengths. Thuswhen a first laser source emits a first wavelength of radiation that isdiscrete from a second wavelength of radiation, it means that no, or avery low level of radiation of the second wavelength is produced by thefirst laser source in a pulse of the first wavelength, for example, lessthan 10% of the intensity at the first wavelength, such as less than 5%,less than 4%, less than 3%, less than 2%, or less than 1%. Typically,when different wavelengths of laser radiation are produced by harmonicsgeneration, or other non-linear frequency conversion processes, thenwhen a specific wavelength is referred to herein, it will be understoodby the skilled person that there will be some degree of variation aboutthe specified wavelength in the spectrum produced by the laser. Forexample, a reference to X nm encompasses a laser producing a spectrum inthe range X±10 nm, such as X±5 nm, for example X±3 nm.

Focusing Optics and Objective Lenses

As matter of routine arrangement, optical components can be used todirect a beam of laser radiation to a focussed spot. FIG. 1 is aschematic diagram of the optics of a prior apparatus set up. Here alaser source (e.g. a pulsed laser source, optionally incorporating apulse picker) 101 emits a beam of laser radiation which is directedthrough an energy control module 102 and then optics 103. The beam ofradiation is then directed towards the sample by beam/illuminationcombining optics 104 through focusing optics and objective lens 105. Thesample is on a sample stage 107 in the sample chamber 106. The samplestage 107 (e.g., a glass slide), may be mounted on a three-axis (i.e. x,y, z) translation stage 108 in the sample chamber 106. The setup of FIG.1 also comprises a camera 111 for viewing the sample using the samefocusing optics and objective lens 105. An illumination source 109 emitsvisible light which is directed to the sample by illumination/inspectionsplitting optics 110, through the beam/illumination combining optics 104and the focusing optics 105.

An alternative arrangement of a prior apparatus set up is shown in FIG.2. Here, a sample 215 is positioned on the opposite side of the samplestage 207 to the objective lens 205 and the laser radiation ablates thesample 215 through the sample stage 207 or through a sample chamber. Insome instances, the sample chamber is held under a vacuum, or a partialvacuum. Ray diagram lines are shown to represent the possible path thelaser beam 216 takes through from the objective lens 205 through theglass slide 207.

As discussed above, one of the main challenges in achieving a spatialresolution of less than 200 nm, for example less than 150 nm, such asless than 100 nm, in traditional IMC and IMS is confining the spot sizeof the laser to less than 200 nm (e.g., less than 150 nm or less than100 nm) in size. The full width at half maximum of the spot size of thelaser is defined by D=0.541λ/NA^(0.91), for systems where numericalaperture (NA) exceeds 0.7, where is λ is the wavelength of the laserlight (used interchangeably with laser radiation herein) and NA is thenumerical aperture of the objective lens 105, 205 of the focusingoptics. Therefore, in traditional IMC and IMS, lasers of a shorterwavelength such as deep UV lasers of wavelength 213 nm, or focusingoptics with an NA above 0.7, e.g. above 0.8, such as high NA (above0.9), are used to reduce the size of the laser spot size and henceimprove resolution.

However, the numerical aperture of a lens is expressed as NA=n×sinθ,where n is the refractive index of the medium between the lens and thesample stage 107, 207 and θ is half the acceptance angle of theobjective. Therefore, maximum theoretical numerical aperture of theobjective lens in typical IMC and IMS (such as the objective lens 105,205 of the prior apparatus set up shown in FIGS. 1 and 2) is limited to1.0 because the refractive index of a vacuum is 1.0 and the refractiveindex of air is around 1.0.

Immersion Lenses

The present invention overcomes the limitations of traditional IMC andIMS by utilising an immersion medium. The immersion medium has arefractive index which is greater than 1.0 and is placed between theobjective lens and the sample stage. In this way, the apparatus of thepresent invention achieves numerical apertures of greater than 1.0 andso the spot size of the laser is less than 200 nm, less than 150 nm, orless than 100 nm. Thus, the present invention provides an apparatus forimaging mass cytometry with spatial resolution of 200 nm or better, 150nm or better, or 100 nm or better.

Accordingly, the invention provides a laser sampling system comprising:

-   -   a sample stage;    -   a laser source; and    -   focusing optics comprising an objective lens, the focusing        optics adapted to direct radiation from the laser source towards        a location on the sample stage; and wherein    -   the laser sampling system further comprises an immersion medium        positioned between the objective lens and the sample stage.

Accordingly, the invention provides an apparatus for analysing abiological sample comprising:

-   -   a sample stage;    -   a laser source; and    -   focusing optics comprising an objective lens, the focusing        optics adapted to direct radiation from the laser source towards        a location on the sample stage; and wherein    -   the apparatus further comprises an immersion medium positioned        between the objective lens and the sample stage.

Typically, the apparatus also includes a mass spectrometry baseddetector.

Accordingly in operation, the sample stage holds the sample, typicallywherein the sample is on a sample carrier and the same stage holds thesample carrier. Laser radiation is then directed through the optics ofthe apparatus, through the objective lens and immersion medium to thesample, where the radiation ablates material from the sample.

In order to achieve the optimal focusing conditions for the laser, theimmersion medium of the present invention has a refractive index ofgreater than 1.00, such as 1.33 or greater, 1.50 or greater, 2.00 orgreater, or 2.50 or greater.

Furthermore, in order to reconstruct the image of a single layer of thethickness (or less than the thickness) of a biological cell or to read athicker specimen layer by layer and generate a 3D image, as discussedfurther herein, the sample preferably has a thickness of 100 micrometersor below, such as 10 micrometers or below, 5 micrometers or below, 2micrometers or below, or 100 nm or below, or 50 nm or below, or 30 nm orbelow. In some embodiments described in more detail herein, thecombination of the objective lens and the immersion medium is referredto as an immersion lens.

Apparatus or sampling systems as described above comprising an immersionmedium in some embodiments comprise a medium or high NA objective lens,such as an NA of 0.7 or more, 0.8 or more, or 0.9 or more. Such systemstypically also comprise a mass detector, such as a TOF mass spectrometrydetector.

Liquid Immersion Medium

In some embodiments of the invention, the immersion medium is a liquidimmersion medium, FIG. 3 is a schematic diagram of the opticsarrangement of an exemplary embodiment of the invention. It containselements in common with the set ups shown in FIGS. 1 and 2. The focusingoptics comprising an objective lens 305 directs a beam of radiation 316from a laser source (source not shown) towards a location on the samplestage 307 and a liquid immersion medium 312 is positioned between theobjective lens 305 and the sample stage 307. Here, a biological sample315 is positioned on the opposite side of the sample stage 307 to theliquid immersion medium 312. Ray diagrams show how the liquid immersionmedium 312 provides tighter focusing conditions than the conventionalset up shown in FIG. 2. Suitable liquids for liquid immersion media arewater which has a refractive index, n, of 1.333, glycerin (n=1.4695), anoil such as paraffin oil (n=1.480), cedarwood oil (n=1.515) andsynthetic oil (n=1.515), or anisole (n=1.5178), bromonaphthalene(n=1.6585) and methylene iodide (n=1.740). Commercial immersion oils arealso available and some of these commercial oils have properties whichare particularly advantageous for application in the present invention.For example, low or non-fluorescing immersion oils such as Olympus LowAuto Fluorescence Immersion Oil and Nikon are particularly useful foruse with short wavelength lasers because they provide improved signal tonoise ratio over general all-purpose immersion oils. Other oils havevery high viscosity, such as Cargille labs type NVH and OVH, which is ofuse when the distance between the objective lens 305 and sample stage307 is large.

Commercial oil-immersion objectives can achieve a maximum numericalaperture of around 1.49 (close to the refractive index of the immersionoil), and are able to focus a 515 nm laser beam to a FWHM focal spotdiameter of around 160 nm.

When a liquid immersion medium is used, the sample needs to bepositioned on the opposite side of the sample carrier to the liquidmedium (as illustrated in FIG. 3) so that a carrier gas can collect theablated material. Accordingly, through-sample carrier ablationtechniques must be applied here. This has the additional benefit of asmaller achievable working distance for the ablation material collectionhardware, and no need to bend the transport conduit between the samplechamber and the detector. This also leads to reductions in the transienttime, thus increasing the achievable ablation rate in spots per second.

Liquid immersion lenses (e.g. objective-in-water or objective-in-oillenses) are commercially available from Olympus, ThorLabs and Leica.

Solid Immersion Medium

In some embodiments of the invention, the invention provides anapparatus wherein the immersion medium is a solid immersion medium. FIG.4 is a schematic diagram of the optics arrangement of further exemplaryembodiment of the invention in which a solid immersion 413 is positionedbetween the objective lens 405 and the sample stage 407. The othercomponents of FIG. 4 correspond to those in FIG. 3.

Similarly to liquid immersion media, the refractive index n of the solidimmersion lens is greater than air. Suitable materials for solidimmersion lenses are glass, such as S-LAH79™ glass type, which has arefractive index of 2.0 when operating with a wavelength of ˜520 nm.Alternative suitable materials for the solid immersion medium of thepresent invention are diamond or fused silica. At 266 nm diamond becomeswell suited for optical applications, since it has a refractive indexof >2.5 in UV it offers an opportunity to focus 266 nm light to the spotsize on the scale of 100 nm or even below that. Fused silica may be moreattractive from a cost perspective. It would be practical both as aspecimen substrate and as a solid immersion lens material. Though, theindex of refraction of fused silica in UV is only ˜1.5 and the spot sizewill be proportionally larger due to that.

There are two standard optical schemes for solid immersion media: thehemispherical immersion lens and the Weierstrass immersion lens.

Hemispherical Solid Immersion Lens:

FIG. 5a ) shows the geometry of a hemispherical solid immersion lens.The hemispherical solid immersion lens is capable of increasing thenumerical aperture of an optical system by the refractive index, n, ofthe material of the lens.

Weierstrass Solid Immersion Lens:

FIG. 5b ) shows the geometry of a Weierstrass solid immersion lens. TheWeierstrass solid immersion lens is a truncated sphere and has a heightfrom the glass slide 507 of (1+1/n)r, where r is the radius of thespherical surface of the lens. The Weierstrass lens is capable ofincreasing the numerical aperture of an optical system by n², hence theWeierstrass lens is capable of further increasing the numerical apertureof an optical system than the hemispherical lens.

Accordingly, the present invention provides an apparatus wherein thesolid immersion medium is a hemispherical solid immersion lens or aWeierstrass solid immersion lens. As shown in FIGS. 3 and 4, when abiological sample 315, 415 is mounted on the sample stage 307, 407, thebiological sample can be mounted on the opposite side of the samplestage to the solid immersion material. The stage on which the sample ismounted can be made of material of the same refractive index as thesolid immersion lens and the solid immersion lens can be made thinner byan amount equal to the thickness of the substrate to maintain the focalspot location.

Combination of Immersion Lenses and Biological Samples

The present invention comprising an immersion medium positioned betweenthe objective lens and the sample stage provide further advantages whenused to analyse a biological sample prepared according to other methodsof the invention described herein (page 112). In particular, the presentinvention provides further advantages when the biological sample has athickness of 100 micrometers or below, such as 10 micrometers or below,or 100 nm or below, or 50 nm or below, or 30 nm or below.

For example, the apparatus of the present invention comprising animmersion medium between the objective lens and the sample stage can beused to analyse a biological sample of thickness of 100 nm or below,such as 50 nm or below, or 30 nm or below. Since the apparatus of thepresent invention provides an apparatus for imaging mass cytometry withspatial resolution of 200 nm or better (preferably 100 nm or less), thespot size of the laser is 200 nm or less (preferably 100 nm or less) andso the ablation depth is typically 200 nm or less (preferably 100 nm orless). Therefore, analysing a biological sample of thickness 200 nm orbelow using the present invention will ablate all the way through thesample (or preferably 100 nm or less, when the laser spot size is 100 nmor less). Thus, the present invention provides the possibility toreconstruct the image of a single layer of the thickness of a biologicalcell by utilising the apparatus with sequential sections of a biologicalcell.

Alternatively, the apparatus of the present invention comprising animmersion medium between the objective lens and the sample stage can beused to analyse a biological sample of 100 micrometers or below, such as10 micrometers or below, 5 micrometers or below, 2 micrometers or below.Sharp focusing of the laser beam by the immersion media as discussedabove creates a very short depth of focus. Hence, the present inventionprovides the opportunity to read a thicker specimen layer by layer andgenerate a 3D image. The skilled person will appreciate that thisanalysis of a thicker specimen will be more straightforward the morehomogenous a sample is. In tissue samples, which are not homogenous,distortions in in the laser light which arise as the laser travelsthrough the tissue and may result in a larger spot size at the focusthan expected.

Accordingly, the invention provides an imaging mass cytometer or imagingmass spectrometer comprising a biological sample, wherein the biologicalsample has a thickness of less than 100 nm, such as less than 50 nm, orless than 30 nm. The invention also provides an imaging mass cytometeror imaging mass spectrometer comprising a biological sample, wherein thebiological sample has a thickness of less than 100 nm, such as less than50 nm, or less than 30 nm, and wherein the imaging mass cytometer orimaging mass spectrometer comprises a solid immersion lens. Theinvention also provides an imaging mass cytometer or imaging massspectrometer comprising a biological sample, wherein the biologicalsample has a thickness of less than 100 nm, such as less than 50 nm, orless than 30 nm, and wherein the imaging mass cytometer or imaging massspectrometer comprises a solid immersion lens.

Alternatives to Immersion Lenses in Combination with Thin BiologicalSamples:

As discussed above, in traditional IMC and IMS, lasers of a shortwavelength such as deep UV lasers of wavelength 213 nm, or focusingoptics with a n NA above 0.6, e.g. above 0.7, e.g. above 0.8, such ashigh NA (above 0.9) are used to reduce the size of the laser spot sizeand hence improve resolution to 100 nm.

The high NA objective may be a refractive optical component and/or areflective optical component. For example, the use of an asphericalmirror can be used allow the reflecting objective to have a numericalaperture of up to 0.99 (Inagawa et al. Scientific Reports 5, 12833(2015). A catadioptric mirror system may also be used. Furthermore, itis possible to provide a short wavelength laser (300 nm or below) byusing lasers of wavelength 266 nm, or 213 nm, 193 nm solid state lasers,or sixth harmonic generation from ND:Yag, ArF, F₂laser, extreme UV lightsuch as 13.5 nm and 7 nm wavelength (CO₂ laser+Sn plasma).

Accordingly, the present invention provides an apparatus for analysing abiological sample comprising:

-   -   a sample stage comprising a first face and a second face, the        first and second faces being opposed, and wherein the first face        is adapted to receive a biological sample; and    -   a laser source; and    -   focusing optics comprising an objective lens, the focusing        optics adapted to direct a beam of radiation from the laser        source towards the second face to a location on the sample        stage; and wherein    -   the objective lens has a numerical aperture of at least 0.7, at        least 0.8, or at least 0.9, such as at least 0.9.

Accordingly, the present invention provides an apparatus for analysing abiological sample comprising:

-   -   a sample stage comprising a first face and a second face, the        first and second faces being opposed, and wherein the first face        is adapted to receive a biological sample; and    -   a laser source; and    -   focusing optics comprising an objective lens, the focusing        optics adapted to direct a beam of radiation from the laser        source towards the second face to a location on the sample        stage; and wherein    -   the laser source has a wavelength of 300 nm or below.

Accordingly, the present invention provides an apparatus for analysing abiological sample comprising:

-   -   a sample stage comprising a first face and a second face, the        first and second faces being opposed, and wherein the first face        is adapted to receive a biological sample; and    -   a laser source, wherein the laser source has a wavelength of 300        nm or below; and    -   focusing optics comprising an objective lens, the focusing        optics adapted to direct a beam of radiation from the laser        source towards the second face to a location on the sample        stage; and wherein    -   the objective lens has a numerical aperture of at least 0.7, at        least 0.8, or at least 0.9, such as at least 0.9.

As described further herein, ablated material may be ionized orre-ionized close to the surface of the sample, for example by laserionization of an ablation plume that has expanded past the point ofsignificant charge neutralization (once ionized/re-ionized). In suchsystems and methods, gas fluidics may not be needed as the plume neednot be delivered to ICP for ionization. Instead, ion optics positionednear the sample may direct ionized labelling atoms directly to a massspectrometer (e.g., a TOF or magnetic sector mass spectrometer). Asdescribed herein, in certain cases, a small ablation spot size mayreduce neutralizaiton and space charge effects for the ions generatedduring ablation, making it easier to both ionize and to direct ions to amass spectrometer. No second pulse for re-ionization is needed in suchcase. The sample stage and immediately surrounding optics (e.g., high NAlens and/or immersion medium) may be operated in a vacuum, or at lowpressure (e.g., to reduce charge through collision) as described furtherherein.

As the skilled person will appreciate, an apparatus may include afocusing optics with objective lens of numeral aperture of at least 0.9and/or a laser source with a wavelength of 300 nm or below. Furthermore,while the high numerical aperture or short wavelength can be used as analternative to or in addition to the immersion lenses as describedherein, the other components of the system remain the same as for theimmersion lens systems.

Scanning System

In certain aspects, ablation of the sample may be performed by ascanning system. A source of radiation, such as a laser beam or acharged particle beam (such as ion beam or electron beam) source may bescanned across a portion of the sample to produce a single transient(e.g., single instance of ablated material). The transient may be anablation plume delivered to ICP for atomization and ionization prior todetection by MS. Alternatively, the transient may be ionized by laserradiation at or close to the surface of the sample, as described herein.Scanning with an ion beam or electron beam (e.g., using ion optics) mayallow for ablation of a small region of interest of the sample at highresolution (such as a single organelle). For example, the region ofinterest may have an area of less than 100,000 nm², less than 50,000nm², or less than 20,000 nm², or less than 10,000 nm². Alternatively,the region of interest could be larger, such as a single cell or even agroup of cells.

In certain embodiments, a laser scanning system directs laser radiationonto the sample to be ablated. As the laser scanner is capable ofredirecting the position of laser focus on the sample much more quicklythan moving the sample stage relative to a stationary laser beam (due tomuch lower or no inertia in the operative components of the scanningsystem), it enables ablation of discrete spots on the sample to beperformed more quickly. This quicker speed can enable a significantlygreater area to be ablated and recorded as a single pixel, or the speedof the laser spot movement can simply translate to, e.g., an increase inpixel acquisition rate, or a combination of both. In addition, the rapidchange in the location of the spot onto which a pulse of laser radiationcan be directed permits the ablation of arbitrary patterns, for instanceso that a whole cell of non-uniform shape is ablated, by a burst ofpulses/shots of laser radiation in rapid succession directed ontolocations on the sample by the laser scanner system, and then ionisedand detected as a single cloud of material, thus enabling single cellanalysis (see the “Sample chamber of the laser ablation sampling system”section at page 33 onwards). A similar rapid-burst technique can also bedeployed in methods using desorption to remove sample material from asample carrier, i.e. cell LIFTing (Laser Induced Forward Transfer).

Accordingly, the invention provides an apparatus for analysing a sample,such as a biological sample, comprising:

-   -   (i) sampling and ionisation system to remove material from the        sample and to ionise said material to form elemental ions,        comprising a laser scanning system and a sample stage;    -   (ii) a detector to receive elemental ions from said sampling and        ionisation system and to detect said elemental ions.

Equally, this combination of features may be combined, as understood asappropriate by the skilled person with any of the other embodimentsdescribed herein. The use of a scanning system to increase theacquisition rate provides numerous advantages over other strategies forincreasing the rate at which a sample is imaged. For instance, an areaof 100 μm×100 μm can be ablated in with a single laser pulse usingappropriately adapted apparatus. However, such ablation results innumerous problems. Ablating a large area of a sample at once with asingle laser pulse leads to the ablated material being broken up intolarge chunks initially flying at velocities near the speed of sound,rather than small particles, and rather than the material beingtransported away quickly from the sample in the flow of carrier gas(described in more detail below), the large chunks may take longer to beentrained (lengthening the washout time of the sample chamber) than thesmaller chunks, fail to be entrained, or just fly randomly off thesample or onto another part of the sample. If the large chunk ofmaterial flies off the sample, any information in that chunk of materialin the form of detectable atoms, such as labelling atoms, is lost. Ifthe chunk of material lands on another part of the sample, informationis lost from the ablated area, and moreover any detectable atoms in thechunk of material now lie on and can interfere with the signal thatwould be acquired from another part of the sample. As differences in thebiological material in an ablated spot (e.g. cartilaginous materialversus muscle) can also affect how the product breaks up, largerablation spots sizes can also compound fractionation of the sample, withsome kinds of material being entrained in the flow of gas to a lesserdegree than others. Furthermore, as described here, in many applicationsa small spot size is preferred, of the order of pm rather than 100s ofμm, and switching between laser spot sizes multiple orders of magnitudedifferent (e.g. 100 μm vs 1 μm) also presents technical challenges. Forinstance, a laser that can ablate with a spot size of 1 μm may not havethe energy to ablate an area with a spot size of 100 μm in a singlelaser pulse, and sophisticated optics are required to facilitate thetransition between 1 μm and 100 μm without significant loss of energy inthe laser beam or loss of sharpness of the ablation spot.

Rather than ablating a 100 μm² single spot, therefore, 100×100 (i.e.10,000) 1 μm diameter spots can be used to ablate the area by rasteringacross the area. A smaller spot size for ablation naturally does notsuffer from the problems described above to such a great extent—theparticles generated by a smaller ablation spot by necessity arethemselves much smaller in size. Furthermore, with smaller spots, theresulting smaller particles resulting from the ablation have shorter andmore defined washout times from the sample chamber. Where each of thesmaller spots is desired to be resolved separately, this in turn has theconsequence that data can be acquired more quickly as the transientsfrom each ablative laser pulse do not overlap when detected in thedetector (or overlap to an acceptable degree, as explained below).

However, moving a sample stage in 1 μm increments along a row, and thendown a row is relatively slow due to inertia as noted above. Thus, byusing a laser scanner system to raster across the area, without movingthe sample stage, or moving the sample stage less frequently or at aconstant speed, the relatively slow speed of the sample stage does notlimit the rate at which the sample can be ablated.

Accordingly, to enable rapid scanning, the laser scanning system must beable to rapidly switch the position at which the laser radiation isbeing directed on the sample. The time taken to switch the ablatingposition of the laser radiation is termed the response time of the laserscanning system. Accordingly, in some embodiments of the invention, theresponse time of the laser sampling system is quicker than 1 ms, quickerthan 500 μs, quicker than 250 μs, quicker than 100 μs, quicker than 50μs, quicker than 10 μs, quicker than 5 μs, quicker than 1 μs, quickerthan 500 ns, quicker than 250 ns, quicker than 100 ns, quicker than 50ns, quicker than 10 ns, or around 1 ns.

The laser scanning system can direct the laser beam in at least onedirection relative to the sample stage on which the sample is positionedduring ablation. In some instances, the laser scanning system can directthe laser radiation in two directions relative to the sample stage. Byway of example, the sample stage may be used to move the sampleincrementally in the X-axis, and the laser may be swept across thesample in the Y axis (see FIGS. 7-9 for illustrations of the relativemovements). When a 1 μm spot size is used, the movement in the X axismay be in 1 μm increments. At a given position in the X axis, the laserscanning system can be used to direct the laser to a series of positions1 μm apart in the Y axis. Because the rate at which the laser scanningsystem can direct the laser radiation to different positions in the Yaxis is much quicker than the stage can move incrementally in the Xaxis, a significant increase in ablation rate is achieved in this simpleillustration of the operation of the scanner.

In some instances, the laser scanning system directs the laser beam inboth the X and Y axes. Accordingly, in this instance more advancedablation patterns can be generated. For instance, when the laserscanning system can direct the laser radiation in both the X and Y axes,the sample stage may be moved at constant speed in the X axis (therebyeliminating inefficiencies associated with the inertia of the samplestage during the movement across each row other thanacceleration/deceleration at the start/end of the row), while the laserscanning system directs laser radiation pulses up and down columns onthe sample whilst compensating for the movement of the sample stage. Toachieve this movement, the triangle-wave control signals can be appliedto the scanner in the X direction, and a sawtooth signal in the Ydirection. Alternatively, it may be desirable to apply a sawtooth drivesignal to the scanner in the Y direction, depending on the processingalgorithm used, as would be appreciated by the skilled person. As afurther alternative, one of the scanner components may be pre-rotatedslightly, to pre-compensate for the slanted scanning pattern. In someembodiments, the controller of the laser scanning system will cause thelaser scanner system to move the beam in a figure-of-eight pattern asthe sample stage moves.

The significantly quicker (re-)direction of laser radiation ontodifferent locations on the sample accordingly enables much quickerablation of large areas of the sample, provided that the laser used inthe laser sampling system has a sufficiently high repetition rate (asdiscussed below). For instance, if only fewer than 5 pulses can bedirected to different locations on a sample per second, the time takento study a 1 mm×1 mm area with ablation at a spot size of 1 μm would beover two days. With a rate of 200 Hz, this would be around 80 minutes,with further reductions in the analysis time for further increases inthe frequency of pulses. However, samples are often significantlylarger. An average microscope slide on which a tissue section can beplaced is 25×75 mm. This would take around 110 days to ablate at a rateof 200 Hz. However, if a laser scanning system is used the time can bedramatically shortened, for instance where the sample stage is moved ata constant speed along the X axis (1 mm/s), while the laser beam ismoved back-and-forth in the Y axis direction with the laser scanningsystem. The laser scanning system can scan the position of the laserfocus at a rate that matches the speed of the stage motion, in thiscase, 500 Hz. This would produce a 1 μm spacing between adjacent linesin the raster pattern at this speed. Then, depending on the maximumlaser repetition rate, the extent of the deflection of the laserradiation by the laser scanning system is chosen to match. Here, toproduce a peak-to-peak amplitude of 100 microns, a 100 kHz laserrepetition rate would be required. This allows the device to process 0.1mm²/s, compared to at most 0.0004 mm²/s for current apparatus. Incomparison to the figure of 110 days discussed above, with a laserscanning system as discussed in this paragraph, it would only takearound 5 hours to process the slide.

Another application is arbitrary ablation area shaping. If a highrepetition rate laser is used, it is possible to deliver a burst ofclosely-spaced laser pulses in the same time that a nanosecond laserwould deliver one pulse. By quickly adjusting the X and Y positions ofthe ablation spot during a burst of laser pulses, ablation craters ofarbitrary shape and size (down to the diffraction limit of the light)can be created. For instance, the n and n+1 positions in a burst may beno more than a distance equal to 10× the laser spot diameter apart(based on the centre of the ablation spot of the nth spot and the(n+1)th spot), such as less than 8×, less than 5×, less than 2.5 times,less than 2× times, less than 1.5×, around 1×, or less than 1× thediameter of the spot size. Particular methods employing this techniqueare discussed in the methods section below, at page 32.

Accordingly, in some embodiments, the laser scanning system comprises apositioner to impart a first relative movement of a laser beam emittedby the laser with respect to the sample stage (e.g. the Y axis relativeto the surface of the sample).

In some embodiments, the positioner of the laser scanning system iscapable of imparting a second relative movement of the laser beam withrespect to the sample stage, wherein the first and second relativemovements are not parallel, such as wherein the relative movements areorthogonal (e.g. the first movement direction is in the Y axis relativeto the surface of the sample and the second movement direction is in theX axis relative to the surface of the sample).

In some embodiments, the laser scanning system further comprises asecond positioner capable of imparting a second relative movement of thelaser beam with respect to the sample stage, wherein the first andsecond relative movements are not parallel, such as wherein the relativemovements are orthogonal (e.g. the first movement direction is in the Yaxis relative to the surface of the sample and the second movementdirection is in the X axis relative to the surface of the sample).

Any component which can rapidly direct laser radiation to differentlocations on the sample can be used as a positioner in the laserscanning system. The various types of positioner discussed below arecommercially available, and can be selected by the skilled person asappropriate for the particular application for which an apparatus is tobe used, as each has inherent strengths and limitations. In someembodiments of the invention, as set out below, multiple of thepositioners discussed below can be combined in a single laser scanningsystem. Positioners can be grouped generally into those that rely onmoving components to introduce relative movements into the laser beam(examples of which include galvanometer mirror, piezoelectric mirror,MEMS mirror, polygon scanner etc.) and those that do not (examples ofwhich include such acousto-optic devices and electro-optic devices). Thetypes of positioners listed in the previous sentence act to controllablydeflect the beam of laser radiation to various angles, which results ina translation of the ablation spot. The laser scanning system maycomprise a single positioner, or may comprise a positioner and a secondpositioner. The description of “positioner” and “second positioner”where two positioners are present in the laser scanning system does notdefine an order in which a pulse of laser radiation hits the positionerson its path from the laser source to the sample.

Galvanometer motors on the shaft of which a mirror is mounted can beused to deflect the laser radiation onto different locations on thesample. Movement can be achieved by using a stationary magnet and amoving coil, or a stationary coil and a moving magnet. The arrangementof a stationary coil and moving magnet produces quicker response times.Typically sensors are present in the motor to sense the position of theshaft and the mirror, thereby providing feedback to the controller ofthe motor. One galvanometer mirror can direct the laser beam within oneaxis, and accordingly pairs of galvanometer mirrors are used to enabledirection of the beam in both X and Y axes using this technology.

One strength of the galvanometer mirror is that it enables large anglesof deflection (much greater than, for example, solid state deflectors),which as a consequence can allow more infrequent movement of the samplestage. However, as the moving components of the motor and the mirrorhave a mass, they will suffer from inertia and so time for accelerationof the components must be accommodated within the sampling method.Typically, non-resonant galvanometer mirrors are used. As will beappreciated by the skilled person, resonant galvanometer mirrors can beused, but an apparatus using only such resonant components aspositioners of the laser scanning system will not be capable ofarbitrary (also known as random access) scanning patterns. As it isbased on a mirror, a galvanometer mirror deflector can degrade laserradiation beam quality and increase the ablation spot size, and so willagain be understood by the skilled person to be most applicable insituations which tolerate such effects on the beam.

Galvanometer-mirror based apparatus can be prone to errors in theirpositioning, through sensor noise or tracking error. Accordingly, insome embodiments, each mirror is associated with a positional sensor,which sensor feeds back on the mirror's position to the galvanometer torefine the position of the mirror. In some instances, the positionalinformation is relayed to another component, such as an AOD or EOD inseries to the galvanometer-mirror, which corrects for mirror positioningerror.

Galvanometer mirror systems and components are commercially availablefrom various manufacturers such as Thorlabs (NJ, USA), Laser2000 (UK),ScanLab (Germany), and Cambridge Technology (MA, USA).

In embodiments comprising only galvanometer mirror based positioners,the rate at which ablative laser pulses are capable of being directed atthe sample may be between 200 Hz-1 MHz, 200 Hz-100 kHz, 200 Hz-50 kHz,200 Hz-10 kHz, 1 kHz-1 MHz, 5 kHz-1 MHz, 10 kHz-1 MHz, 50 kHz-1 MHz, 100kHz-1 MHz, 1 kHz to 100 kHz or 10 kHz-100 kHz.

Accordingly, in some embodiments of the invention, the laser scannersystem comprises one or more positioners which is a galvanometer mirror,such as a galvanometer mirror array.

FIG. 1 is a schematic diagram of the optics of a prior apparatus set up.Here a laser source (e.g. a pulsed laser source, optionallyincorporating a pulse picker) 101 emits a beam of laser radiation whichis directed through an energy control module 102 and then optics 103.The optics 103 may include beam shaping optics. The beam of radiation isthen directed towards the sample by beam/illumination combining optics104 through focusing optics and object lens 105. The sample may be on asupport, such as a glass side 107, sitting on a three-axis (i.e. x, y,z) translation stage 108 in the sample chamber 106. The setup of FIG. 1also comprises a camera 111 for viewing the sample using the samefocusing optics and objective lens 105. An illumination source 109 emitsvisible light which is directed to the sample by illumination/inspectionsplitting optics 110, through the beam/illumination combining optics 104and the focusing optics 105.

In a more advanced configuration, the optics (e.g., optics 103) mayfurther include a positioner (e.g., a movable mirror or scanner asdescribed herein) to enable scanning of the laser across a sample. Forexample, before the beam of laser radiation is shaped by the beamshaping optics of the optics 203, a positioner, such as a galvanometermirror, piezoelectric mirror, MEMS mirror or polygon scanner,\ deflectsthe beam of laser radiation. For example, a single mirror in agalvanometer mirror-based apparatus permits for scanning of the beam inone direction, e.g. the Y axis relative to the sample. The deflectionintroduced by the positioner is carried throughout the optics, resultingin ablation of different locations on the sample dependent on theposition of the mirror. The positioner may be coordinated (e.g., by acontroller) with the position on the sample stage to determine theparticular location on the sample ablated by the beam of laserradiation. The controller may also connects to the laser source tocoordinate the production of laser pulses (so that pulses are producedby the laser source at a time when the positioner is at a definedposition rather than while it is moving between positions).

However, instead of a single mirror positioner, a pair of mirrorpositioners may be used to induce deflections into the beam of laserradiation. As described elsewhere of herein, the mirror pair can bearranged to provide scanning in two orthogonal directions (X and Y),which can compensate for the movement of the sample on the sample stage.

Similarly, piezoelectric actuators on the shaft of which a mirror ismounted can be used as positioners to deflect the laser radiation ontodifferent locations on the sample. Again, as mirror positioners, whichare based on the movement of components with mass, there will inherentlybe inertia and so a time overhead inherent in movement of the mirror bythis component. Accordingly, this positioner will be understood by theskilled person to have application in certain embodiments wherenanosecond response times for the laser scanning system are notmandatory. Similarly, as it is based on a mirror, the piezoelectricmirror positioner may degrade laser radiation beam quality and increasethe ablation spot size, and so will again be understood by the skilledperson to be most applicable in situations which tolerate such effectson the beam.

In piezoelectric mirrors based on a tilt-tip mirror arrangement,direction of the laser radiation onto the sample in the X and Y axes isprovided in a single component.

Piezoelectric mirrors are commercially available from suppliers such asPhysik Instrumente (Germany).

Accordingly, in some embodiments of the invention, the laser scannersystem comprises a piezoelectric mirror, such as a piezoelectric mirrorarray or a tilt-tip mirror.

In embodiments comprising only piezoelectric mirror based positioners,such as a piezoelectric mirror array or a tilt-tip mirror, the rate atwhich ablative laser pulses are capable of being directed at the samplemay be between 200 Hz-1 MHz, 200 Hz-100 kHz, 200 Hz-50 kHz, 200 Hz-10kHz, 1 kHz-1 MHz, 5 kHz-1 MHz, 10 kHz-1 MHz, 50 kHz-1 MHz, 100 kHz-1MHz, 1 kHz to 100 kHz or 10 kHz-100 kHz. A third kind of positionerwhich is dependent on physical movement of the surface directing thelaser radiation onto a sample is a MEMS (Micro-Electro MechanicalSystem) mirror. The micro mirror in this component can be actuated byelectrostatic, electromechanic and piezoelectric effects. A number ofstrengths of this type of component derive from their small size, suchas low weight, ease of positioning in the apparatus and low powerconsumption. However, as deflection of the laser radiation is stillultimately based on the movement of parts in the component, and as suchthe parts will experience inertia. Once again, as it is based on amirror, the MEMS mirror positioner will degrade laser radiation beamquality and increase the ablation spot size, and so the skilled personwill again understand that such scanner components are thereforeapplicable in situations which tolerate such effects on the laserradiation.

MEMS mirrors are commercially available from suppliers such as MirrorcleTechnologies (CA, USA), Hamamatsu (Japan) and Preciseley MicrotechnologyCorporation (Canada).

Accordingly, in some embodiments of the invention, the laser scannersystem comprises a MEMS mirror.

In embodiments comprising only a MEMS mirror based positioner, the rateat which ablative laser pulses are capable of being directed at thesample may be between 200 Hz-1 MHz, 200 Hz-100 kHz, 200 Hz-50 kHz, 200Hz-10 kHz, 1 kHz-1 MHz, 5 kHz-1 MHz, 10 kHz-1 MHz, 50 kHz-1 MHz, 100kHz-1 MHz, 1 kHz to 100 kHz or 10 kHz-100 kHz. A further kind ofpositioner which is dependent on physical movement of the surfacedirecting the laser radiation onto a sample is a polygon scanner. Here,a reflective polygon or multifaceted mirror spins on a mechanical axis,and every time a flat facet of the polygon is traversing the incomingbeam an angular deflected scanning beam is produced. Polygon scannersare one dimensional scanners, can direct the laser beam along a scannedline (and so a secondary positioner is needed in order to introduce asecond relative movement in the laser beam with respect to the sample,or the sample needs to be moved on the sample stage). In contrast to theback-and-forward motion of e.g. a galvanometer based scanner, once theend of one line of the raster scan has been reached, the beam isdirected back to the position at the start of the scan row. The polygonscan be regular or irregular, depending on the application. Spot size isdependent on facet size and flatness, and the scan line length/scanangle on the number of facets. Very high rotational speeds can beachieved, resulting in high scanning speeds. However, this kind ofpositioner does have drawback, in terms of lower positioning/feedbackaccuracy due to facet manufacturing tolerances and axial wobble, as wellas potential wavefront distortion from the mirror surface. The skilledperson will again understand that such scanner components are thereforeapplicable in situations which tolerate such effects on the laserradiation.

Polygon scanners are commercially available for example from PrecisionLaser Scanning (AZ, USA), II-VI (PA, USA), Nidec Copal Electronics Corp(Japan) inter alia.

In embodiments comprising only a polygon scanner based positioner, therate at which ablative laser pulses are capable of being directed at thesample may be between 200 Hz-10 MHz, 200 Hz-1 MHz, 200 Hz-100 kHz, 200Hz-50 kHz, 200 Hz-10 kHz, 1 kHz-10 MHz, 5 kHz-10 MHz, 10 kHz-10 MHz, 50kHz-10 MHz, 100 kHz-10 MHz, 1 kHz-1 MHz, 10 kHz-1 MHz or 100 kHz-1 MHz.Unlike the preceding types for laser scanner system component, EODs aresolid state components—i.e. they comprise no moving parts. Accordingly,they do not experience mechanical inertia in deflecting laser radiationand so have very fast response times, of the order of 1 ns. They also donot suffer from wear as mechanical components do. An EOD is formed of anoptically transparent material (e.g. a crystal) that has a refractiveindex which varies dependent on the electric field applied across it,which in turn is controlled by the application of an electric voltageover the medium. The refraction of the laser radiation is caused by theintroduction of a phase delay across the cross section of the beam. Ifthe refractive index varies linearly with the electric field, thiseffect is referred to as the Pockels effect. If it varies quadraticallywith the field strength, it is referred to as the Kerr effect. The Kerreffect is usually much weaker than the Pockels effect. Two typicalconfigurations are an EOD based on refraction at the interface(s) of anoptical prism, and based on refraction by an index gradient that existsperpendicular to the direction of the propagation of the laserradiation. To place an electric field across the EOD, electrodes arebonded to opposing sides of the optically transparent material that actsas the medium. Bonding one set of opposed electrodes generates a1-dimensional scanning EOD. Bonding a second set of electrodesorthogonally to the first set electrodes generates a 2-dimensional (X,Y) scanner.

The deflection angle of EODs is lower than galvanometer mirrors, forinstance, but by placing several EODs in sequence, the angle can beincreased, if required for a given apparatus set up. Exemplary materialsfor the refractive medium in the EOD include Potassium Tantalate NiobateKTN (KTa_(x)Nb_(1-x)O₃), LiTaO₃, LiNbO₃, BaTiO₃, SrTiO₃, SBN(Sr_(1-x)Ba_(x)Nb₂O₆) and KTiOPO₄ with KTN displaying greater deflectionangles at the same field strength.

The angular accuracy of EODs is high, and is principally dependent onthe accuracy of the driver connected to the electrodes. Further, asnoted above, the response time of EODs is very quick, and quicker eventhan the AODs discussed below (due to the fact that a (changing)electric field in a crystal is established at the speed of light in thematerial, rather than at the acoustic velocity in the material; seediscussion in Romer and Bechtold, 2014, Physics Procedia 56:29-39).

Accordingly, in some embodiments of the invention, the laser scannersystem comprises an EOD. In some embodiments, the EOD is one in whichtwo sets of electrodes have been orthogonally connected to therefractive medium.

In embodiments comprising an EOD based positioner, the rate at whichablative laser pulses are capable of being directed at the sample may bebetween 200 Hz-100 MHz, 200 Hz-10 MHz, 200 Hz-1 MHz, 200 Hz-100 kHz, 200Hz-50 kHz, 200 Hz-10 kHz, 1 kHz-100 MHz, 5 kHz-100 MHz, 10 kHz-100 MHz,50 kHz-100 MHz, 100 kHz-100 MHz, 1 MHz-100 MHz, 10-100 MHz, 1 kHz-10MHz, 10 kHz-10 MHz, or 100 kHz-10 MHz. This class of positioner is alsoa solid state component. The deflection of the component is based onpropagating sound waves in an optically transparent material to induce aperiodically changing refractive index. The changing refractive indexoccurs because of compression and rarefaction of the material (i.e.changing density) due to the sound waves propagating through thematerial. The periodically changing refractive index diffracts a laserbeam traveling through the material by acting like an optical grating.

The AOD is generated by bonding a transducer (typically a piezoelectricelement) to an acousto-optic crystal (e.g. TeO₂). The transducer, drivenby an electrical amplifier, introduces acoustic waves into therefractive medium. At the opposite end, the crystal is typically skewcut and fitted with an acoustic absorbing material to avoid reflectionof the acoustic wave back into the crystal.

As the waves propagate in one direction through the crystal, this formsa 1-dimensional scanner. By placing two AODs orthogonally in series, orby bonding two transducers on orthogonal crystal faces, a 2-dimensionalscanner can be generated.

As for EODs, deflection angle of AODs is lower than galvanometermirrors, but again compared to such mirror-based scanners the angularaccuracy is high, with the frequency driving the crystal being digitallycontrolled, and commonly resolvable to 1 Hz. Römer and Bechtold, 2014,note that drift, common for galvo-based scanners, as well as temperaturedependency in comparison to analog controllers, are not usually problemsencountered by AODs.

Exemplary materials for use as the refractive medium of the AOD includetellurium dioxide, fused silica, crystalline quartz, sapphire, AMTIR,GaP, GaAs, InP, SF6, lithium niobate, PbMoO₄, arsenic trisulfide,tellurite glass, lead silicate, Ge₅₅As₁₂S₃₃, mercury (I) chloride, andlead (II) bromide.

In order to change the angle of deflection, the frequency of soundintroduced to the crystal must be changed, and it takes a finite amountof time for the acoustic wave to fill the crystal (dependent on thespeed of propagation of the soundwave in the crystal and on the size ofthe crystal), thereby meaning there is a degree of delay. Nevertheless,response time is relatively fast, compared to laser system positionersbased on moving parts.

A further characteristic of AODs which can be exploited in particularinstances is that the acoustic power applied to the crystal determineshow much of the laser radiation is diffracted versus the zero-order(i.e. non-diffracted) beam. The non-diffracted beam is typicallydirected to a beam dump. Accordingly, an AOD can be used to effectivelycontrol (or modulate) the intensity and power of the deflected beam athigh speed.

Diffraction efficiency of the AOD is typically non-linear, andaccordingly, curves of diffraction efficiency vs. power can be mappedfor different input frequencies. The mapped efficiency curves for eachfrequency can then be recorded as an equation or in a look-up table forsubsequent use in the apparatus and methods disclosed herein.

Accordingly, in some embodiments of the invention, the laser scannersystem comprises an AOD.

Instead of a rotating mirror a solid state positioner (e.g. an AOD orEOD) may be used to induce deflections into the beam of laser radiationrather than a mirror-based positioner. As described elsewhere of herein,the solid state scanner can scan in two orthogonal directions (X and Y),either by attaching orthogonal electrodes to an EOD medium, or by thearrangement of two AODs in orthogonally in series.

In embodiments comprising an AOD based positioner, the rate at whichablative laser pulses are capable of being directed at the sample may bebetween 200 Hz-100 MHz, 200 Hz-10 MHz, 200 Hz-1 MHz, 200 Hz-100 kHz, 200Hz-50 kHz, 200 Hz-10 kHz, 1 kHz-100 MHz, 5 kHz-100 MHz, 10 kHz-100 MHz,50 kHz-100 MHz, 100 kHz-100 MHz, 1 MHz-100 MHz, 10-100 MHz, 1 kHz-10MHz, 10 kHz-10 MHz, or 100 kHz-10 MHz. In the preceding paragraphs, twotypes of laser scanning system positioners are discussed: mirror based,comprising moving parts, and solid state positioners. The former ischaracterised by high angles of deflection, but comparatively slowresponse times due to inertia. In contrast, solid state positioners havea lower deflection angle range, but much quicker response times.Accordingly, in some embodiments of the invention, the laser scanningsystem includes both mirror based and solid state components in series.This arrangement takes advantages of the strengths of both, e.g. thelarge range provided by the mirror-based components, but accommodatingthe inertia of the mirror-based components. See, for instance, Matsumotoet al., 2013 (Journal of Laser Micro/Nanoengineering 8:315:320).

Accordingly, a solid state positioner AOD or EOD) can be used forinstance to correct for errors in the mirror-based scanner components.In this case, positional sensors relating to mirror-position feedback tothe solid state component, and the angle of deflection introduced intothe beam of laser radiation by the solid state component can be alteredappropriately to correct for positional error of the mirror-basedscanner components.

One example of a combined system includes a galvanometer mirror and anAOD (where the AOD may enable deflection in one or two directions (byusing two AODs in series, or bonding two drivers to orthogonal faces ofthe crystal of a single AOD)). The system may comprise two galvanometermirrors so as to generate a two dimensional scanning system, incombination with an AOD (where the AOD may enable deflection in one ortwo directions (by using two AODs in series, or bonding two drivers toorthogonal faces of the crystal of a single AOD)). In such a system, therate at which ablative laser pulses are capable of being directed at thesample may be between 200 Hz-100 MHz, 200 Hz-10 MHz, 200 Hz-1 MHz, 200Hz-100 kHz, 200 Hz-50 kHz, 200 Hz-10 kHz, 1 kHz-100 MHz, 5 kHz-100 MHz,10 kHz-100 MHz, 50 kHz-100 MHz, 100 kHz-100 MHz, 1 MHz-100 MHz, 10-100MHz, 1 kHz-10 MHz, 10 kHz-10 MHz, or 100 kHz-10 MHz. An alternativeexample of a combined system includes a galvanometer mirror and an EOD(where the EOD may enable deflection in one or two directions (bybonding two orthogonally arranged electrodes to the crystal)). Thesystem may comprise two galvanometer mirrors so as to generate a twodimensional scanning system, in combination with an EOD (where the EODmay enable deflection in one or two directions (by bonding twoorthogonally arranged electrodes to the crystal)). In such as system,the rate at which ablative laser pulses are capable of being directed atthe sample may be between 200 Hz-100 MHz, 200 Hz-10 MHz, 200 Hz-1 MHz,200 Hz-100 kHz, 200 Hz-50 kHz, 200 Hz-10 kHz, 1 kHz-100 MHz, 5 kHz-100MHz, 10 kHz-100 MHz, 50 kHz-100 MHz, 100 kHz-100 MHz, 1 MHz-100 MHz,10-100 MHz, 1 kHz-10 MHz, 10 kHz-10 MHz, or 100 kHz-10 MHz. To controlthe positioners of the laser scanning system, the laser scanning systemmay comprise a scanner control module (such as a computer or aprogrammed chip), which coordinates the movement of the positioners inthe Y and/or X axes, together with the movement of the sample stage. Insome instances, such as back and forth rastering, the appropriatepattern will be pre-programmed into the chip. In other instances,however, inverse kinematics can be applied by the control module todetermine the appropriate ablation pattern to be followed. Inversekinematics may be particularly useful, for example, in generatingarbitrary ablation patterns, so as to plot the best ablation coursebetween multiple and/or irregularly shaped cells to be ablated. Thescanner control module may also co-ordinate the emission of pulses oflaser radiation, e.g. by also co-ordinating operation of the pulsepicker.

Sometimes, a positioner can cause dispersion of the beam of laserradiation it directs. Accordingly, in some embodiments of the apparatusdescribed herein, the laser scanning system comprises at least onedispersion compensator between the positioner and/or the secondpositioner and the sample, adapted so as to compensate for anydispersion caused by the positioner. When the positioner is an AODand/or the second positioner is an AOD the dispersion compensator is (i)a diffraction grating having a line spacing suitable for compensatingfor the dispersion caused by the positioner and/or second positioner;(ii) a prism suitable for compensating for the dispersion caused by thepositioner and/or second positioner appropriate material, thickness, andprism angle); (iii) a combination comprising the diffraction grating (i)and prism (ii); and/or (iv) a further acousto-optic device. In instanceswhere a first positioner causes a dispersion and a second positionercauses a dispersion, the laser scanning system may comprise a firstdispersion compensator to compensate for any dispersion caused by thefirst positioner and a second dispersion compensator to compensate forany dispersion caused by the second positioner. WO03/028940 describeshow another appropriately adapted AOD can be used to compensate fordispersion caused by an AOD positioner.

Sometimes, due to the movement of the positioners directing laserradiation to different locations, the focal length of a beam ofradiation can vary with respect to the position of the sample. This canbe compensated for in a number of ways. For instance, a movable focusinglens can be moved so as to maintain a spot size of constant, or nearconstant, diameter on the sample irrespective of the particular locationon the sample to which the laser radiation is being directed.Alternatively, a tunable focus lens (commercially available fromOptotune), may be used. It is also possible to compensate for spot sizevariation by altering the height of the sample stage in the z axis. Bothof these techniques rely on moving parts, however, introducing a timingoverhead into operation of the system. If an AOD is used with a Gaussianbeam, ablation spot size can be controlled by power applied to thecrystal in the AOD, so as to modulate rapidly first order versus zeroorder beam intensity.

In the alternative arrangements presented in FIGS. 2-4, components maybe similar to those shown in FIG. 1, with the exception that the systemoperates to ablate the sample through the sample carrier. Thisarrangement can be preferred for instance when additional kinetic energyis desired to be imparted into the sample material being ablated, toassist the material's clearance from the area proximal to the ablationspot. Alternatively or in addition, the configurations presented inFIGS. 2, 3 and/or 4 may allow for a smaller spot size as describedherein. In some embodiments, ablation through the sample carrier can becombined with laser scanning optics.

Lasers

Generally, the choice of wavelength and power of the laser used forablation of the sample can follow normal usage in cellular analysis. Thelaser must have sufficient fluence to cause ablation to a desired depth,without substantially ablating the sample carrier. A laser fluence ofbetween 0.1-5 J/cm² is typically suitable e.g. from 3-4 J/cm² or about3.5 J/cm², and the laser will ideally be able to generate a pulse withthis fluence at a rate of 200 Hz or greater. In some instances, a singlelaser pulse from such a laser should be sufficient to ablate cellularmaterial for analysis, such that the laser pulse frequency matches thefrequency with which ablation plumes are generated. In general, to be alaser useful for imaging biological samples, the laser should produce apulse with duration below 100 ns (preferably below 1 ns) which can befocused to, for example, the specific spot sizes discussed herein.

For instance, the frequency of ablation by the laser system is withinthe range 200 Hz-100 MHz, 200 Hz-10 MHz, 200 Hz-1 MHz, 200 Hz-100 kHz,within the range 500-50 kHz, or within the range 1 kHz-10 kHz.

At these frequencies the instrumentation must be able to analyse theablated material rapidly enough to avoid substantial signal overlapbetween consecutive ablations, if it is desired to resolve each ablatedplume individually (which as set out below may not necessarily bedesired when firing a burst of pulses at a sample). It is preferred thatthe overlap between signals originating from consecutive plumes is <10%in intensity, more preferably <5%, and ideally <2%. The time requiredfor analysis of a plume will depend on the washout time of the samplechamber (see sample chamber section below), the transit time of theplume aerosol to and through the laser ionisation system, and the timetaken to analyse the ionised material. Each laser pulse can becorrelated to a pixel on the image of the sample that is subsequentlybuilt up, as discussed in more detail below.

In some embodiments, the laser source comprises a laser with ananosecond pulse duration or an ultrafast laser (pulse duration of 1 μs(10⁻¹² s) or quicker, such as a femtosecond laser. Ultrafast pulsedurations provide a number of advantages, because they limit heatdiffusion from the ablated zone, and thereby provide more precise andreliable ablation craters, as well as minimising scattering of debrisfrom each ablation event. Femtosecond lasers are particularly useful inthe systems and apparatus described here. In particular, femtosecondlasers are highly compatible with systems including laser scanningcomponents, and because the short pulse duration enables ablationtechniques based on multiphoton events and electron seeding processes.Three attributes of femtosecond lasers make them particularly suited forthis application. The first attribute is a high repetition rate oftypical configurations on femtoasecond lasers which is commonly in therange between 1 MHz and 100 MHz. As a contrast, nano-second lasers arecommonly limited in pulse rate below 100 kHz. The second attribute isthe non-linear ablation mechanism. The non-linear ablation sharpens thedefinition of ablation edge and allows for ablation at higher spatialresolution. Thirdly, the non-linear ablation threshold may also be lessmaterial specific which makes it easier to get consistent dimensions forablation spots when studying non-uniform material such as a tissue.

In some instances a femtosecond laser is used as the laser source. Afemtosecond laser is a laser which emits optical pulses with a durationbelow 1 μs. The generation of such short pulses often employs thetechnique of passive mode locking. Femtosecond lasers can be generatedusing a number of types of laser. Typical durations between 30 fs and 30μs can be achieved using passively mode-locked solid-state bulk lasers.Similarly, various diode-pumped lasers, e.g. based on neodymium-doped orytterbium-doped gain media, operate in this regime. Titanium—sapphirelasers with advanced dispersion compensation are even suitable for pulsedurations below 10 fs, in extreme cases down to approximately 5 fs. Thepulse repetition rate is in most cases between 10 MHz and 500 MHz,though there are low repetition rate versions with repetition rates of afew megahertz for higher pulse energies (available from e.g. Lumentum(CA, USA), Radiantis (Spain), Coherent (CA, USA)). This type of lasercan come with an amplifier system which increases the pulse energy

There are also various types of ultrafast fiber lasers, which are alsoin most cases passively mode-locked, typically offering pulse durationsbetween 50 and 500 fs, and repetition rates between 10 and 100 MHz. Suchlasers are commercially available from e.g. NKT Photonics (Denmark;formerly Fianium), Amplitude Systems (France), Laser-Femto (CA, USA).The pulse energy of this type of laser can also be increased by anamplifier, often in the form of an integrated fiber amplifier.

Some mode-locked diode lasers can generate pulses with femtoseconddurations. Directly at the laser output, the pulse duration is usuallyaround several hundred femtoseconds (available e.g. from Coherent (CA,USA)).

Femtosecond lasers are particularly suited for use with immersionlenses, such as oil immersion lenses (as discussed above). Oil immersionlenses can achieve a laser spot diameter of around 160 nm but sinceablation using femtosecond lasers is a multi-photon process, theeffective spot diameter is reduced by a factor of √m when used with afemtosecond laser, where m is the order of the non-linear process. Inthis way, it is possible to achieve an effective spot diameter of lessthan 100 nm. All possible configurations of the apparatus includingimmersion lenses as set out above can achieve a 100 nm ablation spotsize when used with femtosecond lasers, and so the present inventionprovides apparatus and methods which provide a ten times improvement inspatial resolution when compared to traditional IMC and IMS. Femtosecondlasers generally have wavelengths in the near infrared region (700-1300nm), which means frequency-conversion techniques must be employed toshorten their wavelength so that the 100 nm ablation spot diameter canbe achieved. The most straightforward and well-known of these is secondharmonic generation (SHG), which can efficiently convert a near infraredlaser beam to visible wavelengths, where commercial off-the-shelfmicroscope optics can be used.

In some instances, a picosecond laser is used. Many of the types oflasers already discussed in the preceding paragraphs can also be adaptedto produce pulses of picosecond range duration. The most common sourcesare actively or passively mode-locked solid-state bulk lasers, forexample a passively mode-locked Nd-doped YAG, glass or vanadate laser.Likewise, picosecond mode-locked lasers and laser diodes arecommercially available (e.g. NKT Photonics (Denmark), EKSPLA(Lithuania)).

Nanosecond pulse duration lasers (gain switched and Q switched) can alsofind utility in particular apparatus set ups (Coherent (CA, USA),Thorlabs (NJ, USA)). Nanosecond UV lasers are particularly well suitedfor the solid immersion lenses as set out above. In particular,nanosecond UV lasers of 266 nm wavelength can be used with diamond andfused silica solid immersion lenses to focus the light to a spot size of100 nm or below. As one of skill in the art appreciates, laser ablationusing UV lasers is generally a thermal process, which means an areaaround the laser spot size will be affected by the heat from theablation process. This sets limits on the attainable ablation spot size,and so as a result the person of skill in the art accordingly wouldappreciate that UV lasers will be applicable in certain scenarios, butother lasers may be of more utility in other scenarios.

Alternatively, a continuous wave laser may be used, externally modulatedto produce nanosecond or shorter duration pulses.

Typically, the laser beam used for ablation in the laser systemsdiscussed herein has a spot size, i.e., at the sampling location, of 100μm or less, such as 50 μm or less, 25 μm or less, 20 μm or less, 15 μmor less, or 10 μm or less, such as about 3 μm or less, about 2 μm orless, about 1 μm or less, about 500 nm or less, about 250 nm or less.Where an immersion medium is applied between the objective lens and thesample, as described above in the section on page 8, smaller spot sizescan be achieved, such as 200 nm or less, 150 nm or less, or 100 nm orless. The distance referred to as spot size corresponds to the longestinternal dimension of the beam, e.g. for a circular beam it is the beamdiameter, for a square beam it corresponds to the length of the diagonalbetween opposed corners, for a quadrilateral it is the length of thelongest diagonal etc. (as noted above, the diameter of a circular beamwith a Gaussian distribution is defined as the distance between thepoints at which the fluence has decreased to 1/e² times the peakfluence). As an alternative to the Gaussian beam, beam shaping and beammasking can be employed to provide the desired ablation spot. Forexample, in some applications, a square ablation spot with a top hatenergy distribution can be useful (i.e. a beam with near uniform fluenceas opposed to a Gaussian energy distribution). This arrangement reducesthe dependence of the ablation spot size on the ratio between thefluence at the peak of the Gaussian energy distribution and thethreshold fluence. Ablation at close to the threshold fluence providesmore reliable ablation crater generation and controls debris generation.Accordingly, the laser system may comprise beam masking and/or beamshaping components, such as a diffractive optical element, arranged in aGaussian beam to re-shame the beam and produce a laser focal spot ofuniform or near-uniform fluence, such as a fluence that varies acrossthe beam by less than ±25%, such as less than ±20%, ±15%, ±10% or lessthan ±5%. Sometimes, the laser beam has a square cross-sectional shape.Sometimes, the beam has a top hat energy distribution. As set out above,in the context of the present invention, the distance referred to asspot size is the longest internal dimension of the beam at the samplinglocation, i.e. the spot size is the lateral dimension of the beam, sofor example, the spot size of a circular beam is the diameter. However,the skilled person will appreciate that the focal spot of an objectivelens is a three dimensional volume and that the axial dimensions of afocussed spot size are generally longer than the lateral dimensions sothat in some instances, the axial dimension of the focal spot may belonger than the distance referred to as ‘spot size’ in the context ofthe present invention. Beam shaping and masking can also be used toenable an apparatus which can switch between the high resolution spotsizes achievable with immersion lenses to larger spot sizes, if desiredby the user. As well as high resolution imaging under certainconditions, when the project specification requires it, large spots canbe used (e.g. where a lower resolution is suitable, or where a greaterarea of tissue is required to be sampled and analysed in a given time).

When used for analysis of biological samples, in order to analyseindividual cells the spot size of laser beam used will depend on thesize and spacing of the cells. For example, where the cells are tightlypacked against one another (such as in a tissue section) one or morelaser sources in the laser system can have a spot size which is nolarger than these cells. This size will depend on the particular cellsin a sample, but in general the laser spot will have a diameter of lessthan 4 μm e.g. about 3 μm or less, about 2 μm or less, about 1 μm orless, about 500 nm or less, about 250 nm or less. In order to analysegiven cells at a subcellular resolution the system uses a laser spotsize which is no larger than these cells, and more specifically uses alaser spot size which can ablate material with a subcellular resolution.The lower the spot size, the greater the resolution resulting image.Thus, where high resolution subcellular imaging is required, thetechniques described herein can be used. Sometimes, single cell analysiscan be performed using a spot size larger than the size of the cell, forexample where cells are spread out on the slide, with space between thecells (e.g. such as after analysis by electron microscopy such that theinternal structure of the cell has been determined separated from theelemental analysis of the cell). Here, a larger spot size can be usedand single cell characterisation achieved, because the additionalablated area around the cell of interest does not comprise additionalcells. The particular spot size used can therefore be selectedappropriately dependent upon the size of the cells being analysed. Inbiological samples, the cells will rarely all be of the same size, andso if subcellular resolution imaging is desired, the ablation spot sizeshould be smaller than the smallest cell, if constant spot size ismaintained throughout the ablation procedure. Small spot sizes can beachieved using focusing of laser beams. A laser spot diameter of 1 μmcorresponds to a laser focus point (i.e. the diameter of the laser beamat the focal point of the beam) of 1 μm, but the laser focus point canvary by +20% or more due to spatial distribution of energy on the target(for instance, Gaussian beam shape) and variation in total laser energywith respect to the ablation threshold energy. Suitable objectives forfocusing a laser beam include a reflecting objective, such as anobjective of a Schwarzschild Cassegrain design (reverse Cassegrain).Refracting objectives can also be used, as can combinationreflecting-refracting objectives. A single aspheric lens can also beused to achieve the required focusing. A solid-immersion lens ordiffractive optic can also be used to focus the laser beam. Anothermeans for controlling the spot size of the laser, which can be usedalone or in combination with the above objectives is to pass the beamthrough an aperture prior to focusing. Different beam diameters can beachieved by passing the beam through apertures of different diameterfrom an array of diameters. In some instances, there is a singleaperture of variable size, for example when the aperture is a diaphragmaperture. Sometimes, the diaphragm aperture is an iris diaphragm.Variation of the spot size can also be achieved through dithering of theoptics. The one or more lenses and one or more apertures are positionedbetween the laser and the sample stage.

For completeness, the standard lasers for LA at sub-cellular resolution,as known in the art, are excimer or exciplex lasers. Suitable resultscan be obtained using an argon fluoride laser (λ=193 nm). Pulsedurations of 10-15 ns with these lasers can achieve adequate ablationfor certain applications.

Overall, the laser pulse frequency and strength are selected incombination with the response characteristics of the MS detector topermit distinct detection of individual laser ablation plumes. Incombination with using a small laser spot and a sample chamber having ashort washout time, rapid and high resolution imaging is now feasible.

Laser Ablation Focal Point

To maximise the efficiency of a laser to ablate material from a sample,the sample should be at a suitable position with regard to the laser'sfocal point, for example at the focal point, as the focal point is wherethe laser beam will have the smallest diameter and so most concentratedenergy. This can be achieved in a number of ways. A first way is thatthe sample can be moved in the axis of the laser light directed upon it(i.e. up and down the path of the laser light/towards and away from thelaser source) to the desired point at which the light is of sufficientintensity to effect the desired ablation. Alternatively, oradditionally, lenses can be used to move the focal point of the laserlight and so its effective ability to ablate material at the location ofthe sample, for example by demagnification. The one or more lenses arepositioned between the laser and the sample stage. A third way, whichcan be used alone or in combination with either or both of the twopreceding ways, is to alter the position of the laser.

To assist the user of the system in placing the sample at the mostsuitable location for ablation of material from it, a camera can bedirected at the stage holding the sample (discussed in more detailbelow). Accordingly, the disclosure provides a laser ablation samplingsystem comprising a camera directed on the sample stage. The imagedetected by the camera can be focussed to the same point at which thelaser is focussed. This can be accomplished by using the same objectivelens for both laser ablation and optical imaging. By bringing the focalpoint of two into accordance, the user can be sure that laser ablationwill be most effective when the optical image is in focus. Precisemovement of the stage to bring the sample into focus can be effected byuse of piezo activators, as available from Physik Instrumente,Cedrat-technologies, Thorlabs and other suppliers.

In a further mode of operation, the laser ablation is directed to thesample through the sample carrier. In this instance, the sample supportshould be chosen so that it is transparent (at least partially) to thefrequency of laser radiation being employed to ablate the sample.Ablation through the sample can have advantages in particularsituations, because this mode of ablation can impart additional kineticenergy to the plume of material ablated from the sample, driving theablated material further away from the surface of the sample, sofacilitating the ablated material's being transported away from thesample for analysis in the detector.

In order to achieve 3D-imaging of the sample, the sample, or a definedarea thereof, can be ablated to a first depth, which is not completelythrough the sample. Following this, the same area can be ablated againto a second depth, and so on to third, fourth, etc. depths. This way a3D image of the sample can be built up. In some instances, it may bepreferred to ablate all of the area for ablation to a first depth beforeproceeding to ablate at the second depth. Alternatively, repeatedablation at the same spot may be performed to ablate through differentdepths before proceeding onto the next location in the area forablation. In both instances, deconvolution of the resulting signals atthe MS to locations and depths of the sample can be performed by theimaging software. Thick tissue staining can be employed and the tissueis stabilized in the wet state similar to the workflow employed inconfocal imaging (Clendenon et al., 2011. Microsc Microanal.17:614-617).

Sample Chamber of the Laser Ablation Sampling System

The sample is placed in the sample chamber when it is subjected to laserablation. The sample chamber comprises a stage, which holds the sample(typically the sample is on a sample carrier). When ablated, thematerial in the sample forms plumes, and the flow of gas passed throughthe sample chamber from a gas inlet to a gas outlet carries away theplumes of aerosolised material, including any labelling atoms that wereat the ablated location. The gas carries the material to the ionisationsystem, which ionises the material to enable detection by the detector.The atoms, including the labelling atoms, in the sample can bedistinguished by the detector and so their detection reveals thepresence or absence of multiple targets in a plume and so adetermination of what targets were present at the ablated locus on thesample. Accordingly, the sample chamber plays a dual role in hosting thesolid sample that is analysed, but also in being the starting point ofthe transfer of aerosolised material to the ionisation and detectionsystems. This means that the gas flow through the chamber can affect howspread out the ablated plume of material becomes as it passes throughthe system. A measure of how spread out the ablated plume becomes is thewashout time of the sample chamber. This figure is a measure of how longit takes material ablated from the sample to be carried out of thesample chamber by the gas flowing through it.

The spatial resolution of the signals generated from laser ablation(i.e. when ablation is used for imaging rather than exclusively forclearing, as discussed below) in this way depends on factors including:(i) the spot size of the laser, as signal is integrated over the totalarea which is ablated; and the speed with which plumes are generatedversus the movement of the sample relative to the laser, and (ii) thespeed at which a plume can be analysed, relative to the speed at whichplumes are being generated, to avoid overlap of signal from consecutiveplumes as mentioned above. Accordingly, being able to analyse a plume inthe shortest time possible minimises the likelihood of plume overlap(and so in turn enables plumes to be generated more frequently), ifindividual analysis of plumes is desired.

Accordingly, a sample chamber with a short washout time (e.g. 100 ms orless) is advantageous for use with the apparatus and methods disclosedherein. A sample chamber with a long washout time will either limit thespeed at which an image can be generated or will lead to overlap betweensignals originating from consecutive sample spots (e.g. Kindness et al.(2003; Clin Chem 49:1916-23), which had signal duration of over 10seconds). Therefore aerosol washout time is a key limiting factor forachieving high resolution without increasing total scan time. Samplechambers with washout times of ≤100 ms are known in the art. Forexample, Gurevich & Hergenroder (2007; J. Anal. At. Spectrom.,22:1043-1050) discloses a sample chamber with a washout time below 100ms. A sample chamber was disclosed in Wang et al. (2013; Anal. Chem.85:10107-16) (see also WO 2014/146724) which has a washout time of 30 msor less, thereby permitting a high ablation frequency (e.g. above 20 Hz)and thus rapid analysis. Another such sample chamber is disclosed in WO2014/127034. The sample chamber in WO 2014/127034 comprises a samplecapture cell configured to be arranged operably proximate to the target,the sample capture cell including: a capture cavity having an openingformed in a surface of the capture cell, wherein the capture cavity isconfigured to receive, through the opening, target material ejected orgenerated from the laser ablation site and a guide wall exposed withinthe capture cavity and configured to direct a flow of the carrier gaswithin the capture cavity from an inlet to an outlet such that at leasta portion of the target material received within the capture cavity istransferrable into the outlet as a sample. The volume of the capturecavity in the sample chamber of WO 2014/127034 is less than 1 cm³ andcan be below 0.005 cm³. Sometimes the sample chamber has a washout timeof 25 ms or less, such as 20 ms, 10 ms or less, 5 ms or less, 2 ms orless, 1 ms, less or 500 μs or less, 200 μs or less, 100 μs or less, 50μs or less, or 25 μs or less. For example, the sample chamber may have awashout time of 10 μs or more. Typically, the sample chamber has awashout time of 5 ms or less.

For completeness, sometimes the plumes from the sample can be generatedmore frequently than the washout time of the sample chamber, and theresulting images will smear accordingly (e.g. if the highest possibleresolution is not deemed necessary for the particular analysis beingundertaken).

The sample chamber typically comprises a translation stage which holdsthe sample (and sample carrier) and moves the sample relative to a beamof laser radiation. When a mode of operation is used which requires thedirection of laser radiation through the sample carrier to the sample,the stage holding the sample carrier should also be transparent to thelaser radiation used.

Thus, the sample may be positioned on the side of the sample carrier(e.g., glass slide) facing the laser radiation as it is directed ontothe sample, such that ablation plumes are released on, and capturedfrom, the same side as that from which the laser radiation is directedonto the sample. Alternatively, the sample may be positioned on the sideof the sample carrier opposite to the laser radiation as it is directedonto the sample (i.e. the laser radiation passes through the samplecarrier before reaching the sample), and ablation plumes are releasedon, and captured from, the opposite side to the laser radiation.Direction of laser radiation onto the sample through the sample carrieris of particular utility when an immersion medium, such as a solidimmersion lens or a liquid immersion lens.

One feature of a sample chamber, which is of particular use wherespecific portions in various discrete areas of sample are ablated, is awide range of movement in which the sample can be moved in the x and y(i.e. horizontal) axes in relation to the laser (where the laser beam isdirected onto the sample in the z axis), with the x and y axes beingperpendicular to one another. More reliable and accurate relativepositions are achieved by moving the stage within the sample chamber andkeeping the laser's position fixed in the laser ablation sampling systemof the apparatus. The greater the range of movement, the more distantthe discrete ablated areas can be from one another. The sample is movedin relation to the laser by moving the stage on which the sample isplaced. Accordingly, the sample stage can have a range of movementwithin the sample chamber of at least 10 mm in the x and y axes, such as20 mm in the x and y axes, 30 mm in the x and y axes, 40 mm in the x andy axes, 50 mm in the x and y axes, such as 75 mm in the x and y axes.Sometimes, the range of movement is such that it permits the entiresurface of a standard 25 mm by 75 mm microscope slide to be analysedwithin the chamber. Of course, to enable subcellular ablation to beachieved, in addition to a wide range of movement, the movement shouldbe precise. Accordingly, the stage can be configured to move the samplein the x and y axes in increments of less than 10 μm, such as less than5 μm, less than 4 μm, less than 3 μm, less than 2 μm, 1 μm, or less than1 μm, less than 500 nm, less than 200 nm, less than 100 nm. For example,the stage may be configured to move the sample in increments of at least50 nm. Precise stage movements can be in increments of about 1 μm, suchas 1 μm±0.1 μm. Commercially available microscope stages can be used,for example as available from Thorlabs, Prior Scientific, and AppliedScientific Instrumentation. Alternatively, the motorised stage can bebuilt from components, based on positioners providing the desired rangeof movement and suitably fine precision movement, such as the SLC-24positioners from Smaract. The movement speed of the sample stage canalso affect the speed of the analysis. Accordingly, the sample stage hasan operating speed of greater than 1 mm/s, such as 10 mm/s, 50 mm/s or100 mm/s.

Naturally, when a sample stage in a sample chamber has a wide range ofmovement, the sample chamber must be sized appropriately to accommodatethe movements of the stage. Sizing of the sample chamber is thereforedependent on size of the sample to be involved, which in turn determinesthe size of the mobile sample stage. Exemplary sizes of sample chamberhave an internal chamber of 10×10 cm, 15×15 cm or 20×20 cm. The depth ofthe chamber may be 3 cm, 4 cm or 5 cm. The skilled person will be ableto select appropriate dimensions following the teaching herein. Theinternal dimensions of the sample chamber for analysing biologicalsamples using a laser ablation sampler must be bigger than the range ofmovement of the sample stage, for example at least 5 mm, such as atleast 10 mm. This is because if the walls of the chamber are too closeto the edge of the stage, the flow of the carrier gas passing throughthe chamber which takes the ablated plumes of material away from thesample and into the ionisation system can become turbulent. Turbulentflow disturbs the ablated plumes, and so instead of remaining as a tightcloud of ablated material, the plume of material begins to spread outafter it has been ablated and carried away to the ionisation system ofthe apparatus. A broader peak of the ablated material has negativeeffects on the data produced by the ionisation and detection systemsbecause it leads to interference due to peak overlap, and so ultimately,less spatially resolved data, unless the rate of ablation is slowed downto such a rate that it is no longer experimentally of interest.

As noted above, the sample chamber comprises a gas inlet and a gasoutlet that takes material to the ionisation system. However, it maycontain further ports acting as inlets or outlets to direct the flow ofgas in the chamber and/or provide a mix of gases to the chamber, asdetermined to be appropriate by the skilled artisan for the particularablative process being undertaken.

Camera

In addition to identifying the most effective positioning of the samplefor laser ablation, the inclusion of a camera (such as a charged coupleddevice image sensor based (CCD) camera or an active pixel sensor basedcamera), or any other light detecting means in a laser ablation samplingsystem enables various further analyses and techniques. A CCD is a meansfor detecting light and converting it into digital information that canbe used to generate an image. In a CCD image sensor, there are a seriesof capacitors that detect light, and each capacitor represents a pixelon the determined image. These capacitors allow the conversion ofincoming photons into electrical charges. The CCD is then used to readout these charges, and the recorded charges can be converted into animage. An active-pixel sensor (APS) is an image sensor consisting of anintegrated circuit containing an array of pixel sensors, each pixelcontaining a photodetector and an active amplifier, e.g. a CMOS sensor.

A camera can be incorporated into any laser ablation sampling systemdiscussed herein. The camera can be used to scan the sample to identifycells of particular interest or regions of particular interest (forexample cells of a particular morphology), or for fluorescent probesspecific for an antigen, or an intracellular or structure. In certainembodiments, the fluorescent probes are histochemical stains orantibodies that also comprise a detectable metal tag. Once such cellshave been identified, then laser pulses can be directed at theseparticular cells to ablate material for analysis, for example in anautomated (where the system both identifies and ablates thefeature(s)/regions(s), such as cell(s), of interest) or semi-automatedprocess (where the user of the system, for example a clinicalpathologist, identifies the features/region(s) of interest, which thesystem then ablates in an automated fashion). This enables a significantincrease in the speed at which analyses can be conducted, becauseinstead of needing to ablate the entire sample to analyse particularcells, the cells of interest can be specifically ablated. This leads toefficiencies in methods of analysing biological samples in terms of thetime taken to perform the ablation, but in particular in the time takento interpret the data from the ablation, in terms of constructing imagesfrom it. Constructing images from the data is one of the moretime-consuming parts of the imaging procedure, and therefore byminimising the data collected to the data from relevant parts of thesample, the overall speed of analysis is increased.

The camera may record the image from a confocal microscope. Confocalmicroscopy is a form of optical microscopy that offers a number ofadvantages, including the ability to reduce interference from backgroundinformation (light) away from the focal plane. This happens byelimination of out-of-focus light or glare. Confocal microscopy can beused to assess unstained samples for the morphology of the cells, orwhether a cell is a discrete cell or part of a clump of cells. Often,the sample is specifically labelled with fluorescent markers (such as bylabelled antibodies or by labelled nucleic acids). These fluorescentmakers can be used to stain specific cell populations (e.g. expressingcertain genes and/or proteins) or specific morphological features oncells (such as the nucleus, or mitochondria) and when illuminated withan appropriate wavelength of light, these regions of the sample arespecifically identifiable. Some systems described herein therefore cancomprise a laser for exciting fluorophores in the labels used to labelthe sample. Alternatively, an LED light source can be used for excitingthe fluorophores. Non-confocal (e.g. wide field) fluorescent microscopycan also be used to identify certain regions of the biological sample,but with lower resolution than confocal microscopy.

An alternative imaging technique is two-photon excitation microscopy(also referred to as non-linear or multiphoton microscopy). Thetechnique commonly employs near-IR light to excite fluorophores. Twophotons of IR light are absorbed for each excitation event. Scatteringin the tissue is minimized by IR. Further, due to the multiphotonabsorption, the background signal is strongly suppressed. The mostcommonly used fluorophores have excitation spectra in the 400-500 nmrange, whereas the laser used to excite the two-photon fluorescence liesin near-IR range. If the fluorophore absorbs two infrared photonssimultaneously, it will absorb enough energy to be raised into theexcited state. The fluorophore will then emit a single photon with awavelength that depends on the type of fluorophore used that can then bedetected.

When a laser is used to excite fluorophores for fluorescence microscopy,sometimes this laser is the same laser that generates the laser lightused to ablate material from the biological sample, but used at a powerthat is not sufficient to cause ablation of material from the sample.Sometimes the fluorophores are excited by the wavelength of light thatthe laser then ablates the sample with. In others, a differentwavelength may be used, for example by generating different harmonics ofthe laser to obtain light of different wavelengths, or exploitingdifferent harmonics generated in a harmonic generation system, discussedabove, apart from the harmonics which are used to ablate the sample. Forexample, if the fourth and/or fifth harmonic of a Nd:YAG laser are used,the fundamental harmonic, or the second to third harmonics, could beused for fluorescence microscopy.

As an example technique combining fluorescence and laser ablation, it ispossible to label the nuclei of cells in the biological sample with anantibody or nucleic acid conjugated to a fluorescent moiety.Accordingly, by exciting the fluorescent label and then observing andrecording the positions of the fluorescence using a camera, it ispossible to direct the ablating laser specifically to the nuclei, or toareas not including nuclear material. The division of the sample intonuclei and cytoplasmic regions will find particular application in fieldof cytochemistry. By using an image sensor (such as a CCD detector or anactive pixel sensor, e.g. a CMOS sensor), it is possible to entirelyautomate the process of identifying features/regions of interest andthen ablating them, by using a control module (such as a computer or aprogrammed chip) which correlates the location of the fluorescence withthe x,y coordinates of the sample and then directs the ablation laser tothat location. As part of this process the first image taken by theimage sensor may have a low objective lens magnification (low numericalaperture), which permits a large area of the sample to be surveyed.Following this, a switch to an objective with a higher magnification canbe used to home in on the particular features of interest that have beendetermined to fluoresce by higher magnification optical imaging. Thesefeatures recorded to fluoresce may then be ablated by a laser. Using alower numerical aperture lens first has the further advantage that thedepth of field is increased, thus meaning features buried within thesample may be detected with greater sensitivity than screening with ahigher numerical aperture lens from the outset.

In methods and systems in which fluorescent imaging is used, theemission path of fluorescent light from the sample to the camera mayinclude one or more lenses and/or one or more optical filters. Byincluding an optical filter adapted to pass a selected spectralbandwidth from one or more of the fluorescent labels, the system isadapted to handle chromatic aberrations associated with emissions fromthe fluorescent labels. Chromatic aberrations are the result of thefailure of lenses to focus light of different wavelengths to the samefocal point. Accordingly, by including an optical filter, the backgroundin the optical system is reduced, and the resulting optical image is ofhigher resolution. A further way to minimise the amount of emitted lightof undesired wavelengths that reaches the camera is to exploit chromaticaberration of lenses deliberately by using a series of lenses designedfor the transmission and focus of light at the wavelength transmitted bythe optical filter, akin to the system explained in WO 2005/121864.

A higher resolution optical image is advantageous in this coupling ofoptical techniques and laser ablation sampling, because the accuracy ofthe optical image then determines the precision with which the ablatinglaser can be directed to ablate the sample.

Accordingly, in some embodiments disclosed herein, the apparatus of theinvention comprises a camera. This camera can be used on-line toidentify features/areas of the sample, e.g. specific cells, which canthen be sampled, such as by firing a burst of pulses at thefeature/region of interest to ablate sample material from thefeature/region of interest. Where a burst of pulses is directed at thesample, the material in the resulting plumes detected can be as acontinuous event (the plumes from each individual ablation in effectform a single plume, which is then carried on for detection). While eachcloud of sample material formed from the aggregated plumes fromlocations within a feature/region of interest can be analysed together,sample material in plumes from each different feature/region of interestis still kept discrete. That is to say, that sufficient time is leftbetween ablation of different features/areas of interest to allow samplematerial from the nth feature/area interest before ablation of the(n+1)th feature/area is begun.

In a further mode of operation combining both fluorescence analysis andlaser ablation sampling, instead of analysing the entire slide forfluorescence before targeting laser ablation to those locations, it ispossible to fire a pulse from the laser at a spot on the sample (at lowenergy so as only to excite the fluorescent moieties in the samplerather than ablate the sample) and if a fluorescent emission of expectedwavelength is detected, then the sample at the spot can be ablated byfiring the laser at that spot at full energy, and the resulting plumeanalysed by a detector as described below. This has the advantage thatthe rastering mode of analysis is maintained, but the speed isincreased, because it is possible to pulse and test for fluorescence andobtain results immediately from the fluorescence (rather than the timetaken to analyse and interpret ion data from the detector to determineif the region was of interest), again enabling only the loci ofimportance to be targeted for analysis. Accordingly, applying thisstrategy in imaging a biological sample comprising a plurality of cells,the following steps can be performed: (i) labelling a plurality ofdifferent target molecules in the sample with one or more differentlabelling atoms and one or more fluorescent labels, to provide alabelled sample; (ii) illuminating a known location of the sample withlight to excite the one or more fluorescent labels; (iii) observing andrecording whether there is fluorescence at the location; (iv) if thereis fluorescence, directing laser ablation at the location, to form aplume; (v) subjecting the plume to inductively coupled plasma massspectrometry, and (vi) repeating steps (ii)-(v) for one or more furtherknown locations on the sample, whereby detection of labelling atoms inthe plumes permits construction of an image of the sample of the areaswhich have been ablated.

In some instances, the sample, or the sample carrier, may be modified soas to contain optically detectable (e.g., by optical or fluorescentmicroscopy) moieties at specific locations. The fluorescent locationscan then be used to positionally orient the sample in the apparatus. Theuse of such marker locations finds utility, for example, where thesample may have been examined visually “offline”—i.e. in a piece ofapparatus other than the apparatus of the invention. Such an opticalimage can be marked with feature(s)/region(s) of interest, correspondingto particular cells by, say, a physician, before the optical image withthe feature(s)/region(s) of interest highlighted and the sample aretransferred to an apparatus according to the invention. Here, byreference to the marker locations in the annotated optical image, theapparatus of the invention can identify the corresponding fluorescentpositions by use of the camera and calculate an ablative plan for thepositions of the laser pulses accordingly. Accordingly, in someembodiments, the invention comprises an orientation controller modulecapable of performing the above steps.

In some instances, selection of the features/regions of interest mayperformed using the apparatus of the invention, based on an image of thesample taken by the camera of the apparatus of the invention.

Electron Microscope

In some embodiments of the invention, the apparatus also comprisescomponents to perform electron microscopy.

At a general level, an electron microscope comprises an electron gun(e.g. with a tungsten filament cathode), andelectrostatic/electromagnetic lenses and apertures that control the beamto direct it onto a sample in a sample chamber. The sample is held undervacuum, so that gas molecules cannot impede or diffract electrons ontheir way from the electron gun to the sample. In transmission electronmicroscopy (TEM), the electrons pass through the sample, whereupon theyare deflected. The deflected electrons are then detected by a detectorsuch as a fluorescent screen, or in some instances a high-resolutionphosphor coupled to a CCD. Between the sample and the detector is anobjective lens which controls the magnification of the deflectedelectrons on the detector.

TEM requires ultrathin sections to enable sufficient electrons to passthrough the sample such that an image may be reconstructed from thedeflected electrons that hit the detector. Typically, TEM samples are100 nm or thinner, as prepared by use of an ultramicrotome. Biologicaltissue specimens are chemically fixed, dehydrated and embedded in apolymer resin to stabilize them sufficiently to allow the ultrathinsectioning. Sections of biological specimens, organic polymers andsimilar materials may require staining with heavy atom labels in orderto achieve the required image contrast, as unstained biological samplesin their native unstained state rarely interact strongly with electrons,so as to deflect them to allow electron microscopy images to berecorded.

As noted above, when thin sections are used, it is possible to performelectron microscopy on a sample also analysed by IMS or IMC.Accordingly, high resolution structural images can be obtained byelectron microscopy, for example transmission electron microscopy, andthen this high resolution image used to refine the resolution of imagedata obtained by IMS or IMC to a resolution beyond that achievable withablation using laser radiation (due to the much shorter wavelength ofelectrons compared to photons). In some instances, both electronmicroscopy and elemental analysis by IMC or IMS are performed on thesample in a single apparatus (as IMC/IMS are destructive processes,electron microscopy is performed prior to IMC/IMS)

Thus, the invention provides an imaging mass cytometer or imaging massspectrometer as described herein further comprising an electronmicroscope, such as comprising components as set out above, e.g. anelectron source, such as an electron gun. As will be understood by oneof skill in the art, the particular arrangement of the components willvary (e.g. direction from which electrons are directed onto the sampleand the direction from which laser radiation is directed onto thesample), and routine arrangement of components can be achieved withoutundue burden. In some instances, the sample is not moved within theapparatus between analysis by electron microscopy and subsequentablation. As the person of skill in the art will understand, electronmicroscopy is performed under a vacuum, but ablation as discussed inthis section is performed in the presence of a flow of gas that entrainsparticulate material in the plume generated by ablation of the sample.Accordingly, after completion of the electron microscopy stage ofanalysis, the sample chamber will be allowed to return to closer toatmospheric pressure before elemental analysis is performed.

In ICP and IMS apparatus comprising an electron microscope, thearrangement of components may be such that laser radiation for ablationis directed to the sample through the sample carrier, e.g. as in FIG. 3or 4, the sample carrier can act as part of the wall of the samplechamber, allowing the sample chamber to be kept under vacuum, forelectron microscopy purposes. Accordingly, in some embodiments herein,the apparatus comprises an electron microscope and an immersion medium,such as a liquid immersion lens or a solid immersion lens.

Laser Ablation

Laser ablation may be performed in a manner as set out previously, forexample in Giesen et al, 2014 and WO2014169394, in light of themodifications related herein (e.g. it is not mandatory to use an ICP toionize the sample material, nor to use a TOF MS detector). For example,methods and systems for ionization at or near the sample surface, asdescribed herein, may use ion optics to transfer labelling atoms to amass spectrometry detector (e.g., a TOF detector or magnetic sectordetector) directly from the sample, without the need of gas fluidics todeliver sample to an ICP. In some cases, methods and systems may usenon-laser forms of radiation (e.g., an electron beam, or ion beam)instead of, or in addition to, a laser.

In embodiments where laser ablation is performed without sustainedionization of the ablated sample, the ablation plume may be transferredto an ICP-MS as described below.

Transfer Conduit

The transfer conduit forms a link between the laser ablation samplingsystem and the ionisation system, and allows the transportation ofplumes of sample material, generated by the laser ablation of thesample, from the laser ablation sampling system to the ionisationsystem. Part (or all) of the transfer conduit may be formed, forexample, by drilling through a suitable material to produce a lumen(e.g., a lumen with a circular, rectangular or other cross-section) fortransit of the plume. The transfer conduit sometimes has an innerdiameter in the range 0.2 mm to 3 mm. Sometimes, the internal diameterof the transfer conduit can be varied along its length. For example, thetransfer conduit may be tapered at an end. A transfer conduit sometimeshas a length in the range of 1 centimeter to 100 centimeters. Sometimesthe length is no more than 10 centimeters (e.g., 1-10 centimeters), nomore than 5 centimeters (e.g., 1-5 centimeters), or no more than 3 cm(e.g., 0.1-3 centimeters). Sometimes the transfer conduit lumen isstraight along the entire distance, or nearly the entire distance, fromthe ablation system to the ionisation system. Other times the transferconduit lumen is not straight for the entire distance and changesorientation. For example, the transfer conduit may make a gradual 90degree turn. This configuration allows for the plume generated byablation of a sample in the laser ablation sampling system to move in avertical plane initially while the axis at the transfer conduit inletwill be pointing straight up, and move horizontally as it approaches theionisation system (e.g. an ICP torch which is commonly orientedhorizontally to take advantage of convectional cooling). The transferconduit can be straight for a distance of least 0.1 centimeters, atleast 0.5 centimeters or at least 1 centimeter from the inlet aperturethough which the plume enters or is formed. In general terms, typically,the transfer conduit is adapted to minimize the time it takes totransfer material from the laser ablation sampling system to theionisation system.

Transfer Conduit Inlet, Including Sample Cone

The transfer conduit comprises an inlet in the laser ablation samplingsystem (in particular within the sample chamber of the laser ablationsampling system; it therefore also represents the principal gas outletof the sample chamber). The inlet of the transfer conduit receivessample material ablated from a sample in the laser ablation samplingsystem, and transfers it to the ionisation system. In some instances,the laser ablation sampling system inlet is the source of all gas flowalong the transfer conduit to the ionisation system. In some instances,the laser ablation sampling system inlet that receives material from thelaser ablation sampling system is an aperture in the wall of a conduitalong which a second “transfer” gas is flowed (as disclosed, for examplein WO2014146724 and WO2014147260) from a separate transfer flow inlet.In this instance, the transfer gas forms a significant proportion, andin many instances the majority of the gas flow to the ionisation system.The sample chamber of the laser ablation sampling system contains a gasinlet. Flowing gas into the chamber through this inlet creates a flow ofgas out of the chamber though the inlet of the transfer conduit. Thisflow of gas captures plumes of ablated material, and entrains it as itinto the transfer conduit (typically the laser ablation sampling systeminlet of the transfer conduit is in the shape of a cone, termed hereinthe sample cone) and out of the sample chamber into the conduit passingabove the chamber. This conduit also has gas flowing into it from theseparate transfer flow inlet (left hand side of the figure, indicated bythe transfer flow arrow). The component comprising the transfer flowinlet, laser ablation sampling system inlet and which begins thetransfer conduit which carries the ablated sample material towards theionisation system can also termed a flow cell (as it is in WO2014146724and WO2014147260).

The transfer flow fulfils at least three roles: it flushes the plumeentering the transfer conduit in the direction of the ionisation system,and prevents the plume material from contacting the side walls of thetransfer conduit; it forms a “protection region” above the samplesurface and ensures that the ablation is carried out under a controlledatmosphere; and it increases the flow speed in the transfer conduit.Usually, the viscosity of the capture gas is lower than the viscosity ofthe transfer gas. This helps to confine the plume of sample material inthe capture gas in the center of the transfer conduit and to minimizethe diffusion of the plume of sample material downstream of the laserablation sampling system (because in the center of the flow, thetransport rate is more constant and nearly flat). The gas(es) may be,for example, and without limitation, argon, xenon, helium, nitrogen, ormixtures of these. A common transfer gas is argon. Argon is particularlywell-suited for stopping the diffusion of the plume before it reachesthe walls of the transfer conduit (and it also assists improvedinstrumental sensitivity in apparatus where the ionisation system is anargon gas-based ICP). The capture gas is preferably helium. However, thecapture gas may be replaced by or contain other gases, e.g., hydrogen,nitrogen, or water vapor. At 25° C., argon has a viscosity of 22.6 μPas,whereas helium has a viscosity of 19.8 μPas. Sometimes, the capture gasis helium and the transfer gas is argon.

As described in WO2014169394, the use of a sample cone minimizes thedistance between the target and the laser ablation sampling system inletof the transfer conduit. Because of the reduced distance between thesample and the point of the cone through which the capture gas can flowcone, this leads to improved capture of sample material with lessturbulence, and so reduced spreading of the plumes of ablated samplematerial. The inlet of the transfer conduit is therefore the aperture atthe tip of the sample cone. The cone projects into the sample chamber.

An optional modification of the sample cone is to make it asymmetrical.When the cone is symmetrical, then right at the center the gas flow fromall directions neutralizes, so the overall flow of gas is zero along thesurface of the sample at the axis of the sample cone. By making the coneasymmetrical, a non-zero velocity along the sample surface is created,which assists in the washout of plume materials from the sample chamberof the laser ablation sampling system.

In practice, any modification of the sample cone that causes a non-zerovector gas flow along the surface of the sample at the axis of the conemay be employed. For instance, the asymmetric cone may comprise a notchor a series of notches, adapted to generate non-zero vector gas flowalong the surface of the sample at the axis of the cone. The asymmetriccone may comprise an orifice in the side of the cone, adapted togenerate non-zero vector gas flow along the surface of the sample at theaxis of the cone. This orifice will imbalance gas flows around the cone,thereby again generating a non-zero vector gas flow along the surface ofthe sample at the axis of the cone at the target. The side of the conemay comprise more than one orifice and may include both one or morenotches and one or more orifices. The edges of the notch(es) and/ororifice(s) are typically smoothed, rounded or chamfered in order toprevent or minimize turbulence.

Different orientations of the asymmetry of the cone will be appropriatefor different situations, dependent on the choice of capture andtransfer gas and flow rates thereof, and it is within the abilities ofthe skilled person to appropriately identify the combinations of gas andflow rate for each orientation.

All of the above adaptations may be present in a single asymmetricsample cone as use in the invention. For example, the cone may beasymmetrically truncated and formed from two different elliptical conehalves, the cone may be asymmetrically truncated and comprise one ofmore orifices and so on.

The sample cone is therefore adapted to capture a plume of materialablated from a sample in the laser ablation sampling system. In use, thesample cone is positioned operably proximate to the sample, e.g. bymanoeuvring the sample within the laser ablation sampling system on amovable sample carrier tray, as described already above. As noted above,plumes of ablated sample material enter the transfer conduit through anaperture at the narrow end of the sample cone. The diameter of theaperture can be a) adjustable; b) sized to prevent perturbation to theablated plume as it passes into the transfer conduit; and/or c) aboutthe equal to the cross-sectional diameter of the ablated plume.

Tapered Conduits

In tubes with a smaller internal diameter, the same flow rate of gasmoves at a higher speed. Accordingly, by using a tube with a smallerinternal diameter, a plume of ablated sample material carried in the gasflow can be transported across a defined distance more rapidly at agiven flow rate (e.g. from the laser ablation sampling system to theionisation system in the transfer conduit). One of the key factors inhow quickly an individual plume can be analysed is how much the plumehas diffused during the time from its generation by ablation through tothe time its component ions are detected at the mass spectrometercomponent of the apparatus (the transience time at the detector).Accordingly, by using a narrow transfer conduit, the time betweenablation and detection is reduced, thereby meaning diffusion isdecreased because there is less time in which it can occur, with theultimate result that the transience time of each ablation plume at thedetector is reduced. Lower transience times mean that more plumes can begenerated and analyzed per unit time, thus producing images of higherquality and/or faster.

The taper may comprise a gradual change in the internal diameter of thetransfer conduit along said portion of the length of the transferconduit (i.e. the internal diameter of the tube were a cross sectiontaken through it decreases along the portion from the end of the portiontowards the inlet (at the laser ablation sampling system end) to theoutlet (at the ionisation system end). Usually, the region of theconduit near where ablation occurs has a relatively wide internaldiameter. The larger volume of the conduit before the taper facilitatesthe confinement of the materials generated by ablation. When the ablatedparticles fly off from the ablated spot they travel at high velocities.The friction in the gas slows these particles down but the plume canstill spread on a sub-millimeter to a millimeter scale. Allowing forsufficient distances to the walls helps with the containment of theplume near the center of the flow.

Because the wide internal diameter section is only short (of the orderof 1-2 mm), it does not contribute significantly to the overalltransience time providing the plume spends more time in the longerportion of the transfer conduit with a narrower internal diameter. Thus,a larger internal diameter portion is used to capture the ablationproduct and a smaller internal diameter conduit is used to transportthese particles rapidly to the ionisation system.

The diameter of the narrow internal diameter section is limited by thediameter corresponding to the onset of turbulence. A Reynolds number canbe calculated for a round tube and a known flow. In general a Reynoldsnumber above 4000 will indicate a turbulent flow, and thus should beavoided. A Reynolds number above 2000 will indicate a transitional flow(between non-turbulent and turbulent flow), and thus may also be desiredto be avoided. For a given mass flow of gas the Reynolds number isinversely proportional to the diameter of the conduit. The internaldiameter of the narrow internal diameter section of the transfer conduitcommonly is narrower than 2 mm, for example narrower than 1.5 mm,narrower than 1.25 mm, narrower than 1 mm, but greater than the diameterat which a flow of helium at 4 liters per minute in the conduit has aReynolds number greater than 4000.

Rough or even angular edges in the transitions between the constantdiameter portions of the transfer conduit and the taper may causeturbulence in the gas flow, and typically are avoided.

Sacrificial Flow

At higher flows, the risk of turbulence occurring in the conduitincreases. This is particularly the case where the transfer conduit hasa small internal diameter (e.g. 1 mm). However, it is possible toachieve high speed transfer (up to and in excess of 300 m/s) in transferconduits with a small internal diameter if a light gas, such as heliumor hydrogen, is used instead of argon which is traditionally used as thetransfer flow of gas.

High speed transfer presents problems insofar as it may cause the plumesof ablated sample material to be passed through the ionisation systemwithout an acceptable level of ionisation occurring. The level ofionisation can drop because the increased flow of cool gas reduces thetemperature of the plasma at the end of the torch. If a plume of samplematerial is not ionised to a suitable level, information is lost fromthe ablated sample material—because its components (including anylabelling atoms/elemental tags) cannot be detected by the massspectrometer. For example, the sample may pass so quickly through theplasma at the end of the torch in an ICP ionisation system that theplasma ions do not have sufficient time to act on the sample material toionise it. This problem, caused by high flow, high speed transfer innarrow internal diameter transfer conduits can be solved by theintroduction of a flow sacrificing system at the outlet of the transferconduit. The flow sacrificing system is adapted to receive the flow ofgas from the transfer conduit, and pass only a portion of that flow (thecentral portion of the flow comprising any plumes of ablated samplematerial) onwards into the injector that leads to the ionisation system.To facilitate dispersion of gas from the transfer conduit in the flowsacrificing system, the transfer conduit outlet can be flared out.

The flow sacrificing system is positioned close to the ionisationsystem, so that the length of the tube (e.g. injector) that leads fromthe flow sacrificing system to the ionisation system is short (e.g. ˜1cm long; compared to the length of the transfer conduit which is usuallyof a length of the order of tens of cm, such as ˜50 cm). Thus the lowergas velocity within the tube leading from the flow sacrificing system tothe ionisation system does not significantly affect the total transfertime, as the relatively slower portion of the overall transport systemis much shorter.

In most arrangements, it is not desirable, or in some cases possible, tosignificantly increase the diameter of the tube (e.g. the injector)which passes from the flow sacrificing system to the ionisation systemas a way of reducing the speed of the gas at a volumetric flow rate. Forexample, where the ionisation system is an ICP, the conduit from theflow sacrificing system forms the injector tube in the center of the ICPtorch. When a wider internal diameter injector is used, there is areduction in signal quality, because the plumes of ablated samplematerial cannot be injected so precisely into the center of the plasma(which is the hottest and so the most efficiently ionising part of theplasma). The strong preference is for injectors of 1 mm internaldiameter, or even narrower (e.g. an internal diameter of 800 μm or less,such as 600 μm or less, 500 μm or less or 400 μm or less). Otherionisation techniques rely on the material to be ionised within arelatively small volume in three dimensional space (because thenecessary energy density for ionisation can only be achieved in a smallvolume), and so a conduit with a wider internal diameter means that muchof the sample material passing through the conduit is outside of thezone in which energy density is sufficient to ionise the samplematerial. Thus narrow diameter tubes from the flow sacrificing systeminto the ionisation system are also employed in apparatus with non-ICPionisation systems. As noted above, if a plume of sample material is notionised to a suitable level, information is lost from the ablated samplematerial—because its components (including any labelling atoms/elementaltags) cannot be detected by the mass spectrometer.

Pumping can be used to help ensure a desired split ratio between thesacrificial flow and the flow passing into the inlet of the ionisationsystem. Accordingly, sometimes, the flow sacrificing system comprises apump attached to the sacrificial flow outlet. A controlled restrictorcan be added to the pump to control the sacrificial flow. Sometimes, theflow sacrificing system also comprises a mass flow controller, adaptedto control the restrictor.

Where expensive gases are used, the gas pumped out of the sacrificialflow outlet can be cleaned up and recycled back into the same systemusing known methods of gas purification. Helium is particularly suitedas a transport gas as noted above, but it is expensive; thus, it isadvantageous to reduce the loss of helium in the system (i.e. when it ispassed into the ionisation system and ionised). Accordingly, sometimes agas purification system is connected to the sacrificial flow outlet ofthe flow sacrificing system.

Ionisation System

In order to generate elemental ions, it is necessary to use a hardionisation technique that is capable of vaporising, atomising andionising the atomised sample.

Inductively Coupled Plasma Torch

Commonly, an inductively coupled plasma is used to ionise the materialto be analysed before it is passed to the mass detector for analysis. Itis a plasma source in which the energy is supplied by electric currentsproduced by electromagnetic induction. The inductively coupled plasma issustained in a torch that consists of three concentric tubes, theinnermost tube being known as the injector.

The induction coil that provides the electromagnetic energy thatmaintains the plasma is located around the output end of the torch. Thealternating electromagnetic field reverses polarity many millions oftimes per second. Argon gas is supplied between the two outermostconcentric tubes. Free electrons are introduced through an electricaldischarge and are then accelerated in the alternating electromagneticfield whereupon they collide with the argon atoms and ionise them. Atsteady state, the plasma consists of mostly of argon atoms with a smallfraction of free electrons and argon ions.

The ICP can be retained in the torch because the flow of gas between thetwo outermost tubes keeps the plasma away from the walls of the torch. Asecond flow of argon introduced between the injector (the central tube)and the intermediate tube keeps the plasma clear of the injector. Athird flow of gas is introduced into the injector in the centre of thetorch. Samples to be analysed are introduced through the injector intothe plasma.

The ICP can comprise an injector with an internal diameter of less than2 mm and more than 250 μm for introducing material from the sample intothe plasma. The diameter of the injector refers to the internal diameterof the injector at the end proximal to the plasma. Extending away fromthe plasma, the injector may be of a different diameter, for example awider diameter, wherein the difference in diameter is achieved through astepped increase in diameter or because the injector is tapered alongits length. For instance, the internal diameter of the injector can bebetween 1.75 mm and 250 μm, such as between 1.5 mm and 300 μm indiameter, between 1.25 mm and 300 μm in diameter, between 1 mm and 300μm in diameter, between 900 μm and 300 μm in diameter, between 900 μmand 400 μm in diameter, for example around 850 μm in diameter. The useof an injector with an internal diameter less than 2 mm providessignificant advantages over injectors with a larger diameter. Oneadvantage of this feature is that the transience of the signal detectedin the mass detector when a plume of sample material is introduced intothe plasma is reduced with a narrower injector (the plume of samplematerial being the cloud of particular and vaporous material removedfrom the sample by the laser ablation sampling system). Accordingly, thetime taken to analyse a plume of sample material from its introductioninto the ICP for ionisation until the detection of the resulting ions inthe mass detector is reduced. This decrease in time taken to analyse aplume of sample material enables more plumes of sample material to bedetected in any given time period. Also, an injector with a smallerinternal diameter results in the more accurate introduction of samplematerial into the centre of the induction coupled plasma, where moreefficient ionisation occurs (in contrast to a larger diameter injectorwhich could introduce sample material more towards the fringe of theplasma, where ionisation is not as efficient). ICP torches (Agilent,Varian, Nu Instruments, Spectro, Leeman Labs, PerkinElmer, Thermo Fisheretc.) and injectors (for example from Elemental Scientific and Meinhard)are available.

Other Ionisation Techniques

Electron Ionisation

Electron ionisation involves bombarding a gas-phase sample with a beamof electrons. An electron ionisation chamber includes a source ofelectrons and an electron trap. A typical source of the beam ofelectrons is a rhenium or tungsten wire, usually operated at 70 electronvolts energy. Electron beam sources for electron ionisation areavailable from Markes International. The beam of electrons is directedtowards the electron trap, and a magnetic field applied parallel to thedirection of the electrons travel causes the electrons to travel in ahelical path. The gas-phase sample is directed through the electronionisation chamber and interacts with the beam of electrons to formions. Electron ionisation is considered a hard method of ionisationsince the process typically causes the sample molecules to fragment.Examples of commercially available electron ionisation systems includethe Advanced Markus Electron Ionisation Chamber.

Optional Further Components of the Laser Ablation Based Sampling andIonisation System Ion Deflector

Mass spectrometers detect ions when they hit a surface of theirdetector. The collision of an ion with the detector causes the releaseof electrons from the detector surface. These electrons are multipliedas they pass through the detector (the first released electron knocksout further electrons in the detector, these electrons then hitsecondary plates which further amplify the number of electrons). Thenumber of electrons hitting the anode of the detector generates acurrent. The number of electrons hitting the anode can be controlled byaltering the voltage applied to the secondary plates. The current is ananalog signal that can then be converted into a count of the ionshitting the detector by an analog-digital converter. When the detectoris operating in its linear range, the current can be directly correlatedto the number of ions. The quantity of ions that can be detected at oncehas a limit (which can be expressed as the number of ions detectable persecond). Above this point, the number electrons released by ions hittingthe detector is no longer correlated to the number of ions. Thistherefore places an upper limit on the quantitative capabilities of thedetector.

When ions hit the detector, its surface becomes damaged bycontamination. Over time, this irreversible contamination damage resultsin fewer electrons being released by the detector surface when an ionhits the detector, with the ultimate result that the detector needsreplacing. This is termed “detector aging”, and is a well-knownphenomenon in MS.

Detector life can therefore be lengthened by avoiding the introductionof overloading quantities of ions into the MS. As noted above, when thetotal number of ions hitting the MS detector exceeds the upper limit ofdetection, the signal is not as informative as when the number of ionsis below the upper limit because it is no longer quantitative. It istherefore desirable to avoid exceeding the upper limit of detection asit results in accelerated detector aging without generating useful data.

Analysis of large packets of ions by mass spectrometry involves aparticular set of challenges not found in normal mass spectrometry. Inparticular, typical MS techniques involve introducing a low and constantlevel of material into the detector, which should not approach the upperdetection limit or cause accelerated aging of the detector. On the otherhand, laser ablation-based techniques analyse a relatively large amountof material in a very short time window in the MS: e.g. the ions from acell-sized patch of a tissue sample which is much larger than the smallpackets of ions typically analysed in MS. In effect, it is a deliberatealmost overloading of the detector with analysed packed of ionsresulting from ablation or lifting. In between the analysis events thesignal is at baseline (a signal that is close to zero because no ionsfrom labelling atoms are deliberately being entering into the MS fromthe sampling and ionisation system; some ions will inevitably bedetected because the MS is not a complete vacuum).

Thus in apparatus described herein, there is an elevated risk ofaccelerated detector aging, because the ions from packets of ionisedsample material labelled with a large number of detectable atoms canexceed the upper limit of detection and damage the detector withoutproviding useful data.

To address these issues, the apparatus can comprise an ion deflectorpositioned between the sampling and ionisation system and the detectorsystem (a mass spectrometer), operable to control the entry of ions intothe mass spectrometer. In one arrangement, when the ion deflector is on,the ions received from the sampling and ionisation system are deflected(i.e. the path of the ions is changed and so they do not reach thedetector), but when the deflector is off the ions are not deflected andreach the detector. How the ion deflector is deployed will depend on thearrangement of the sampling and ionisation system and MS of theapparatus. E.g. if the portal through which the ions enter the MS is notdirectly in line with the path of ions exiting the sampling andionisation system, then by default the appropriately arranged iondeflector will be on, in order to direct ions from the sampling andionisation system into the MS. When an event resulting from theionisation a packet of ionised sample material considered likely tooverload the MS is detected (see below), the ion deflector is switchedoff, so that the rest of the ionised material from the event is notdeflected into the MS and can instead simply hit an internal surface ofthe system, thereby preserving the life of the MS detector. The iondeflector is returned to its original state after the ions from thedamaging event have been prevented from entering the MS, therebyallowing the ions from subsequent packets of ionised sample material toenter the MS and be detected.

Alternatively, in arrangements where (under normal operating conditions)there is no change in the direction of the ions emerging from thesampling and ionisation system before they enter the MS the iondeflector will be off, and the ions from the sampling and ionisationsystem will pass through it to be analysed in the MS. To prevent damagewhen a potential overload of the detector is detected, in thisconfiguration the ion deflector is turned on, and so diverts ions sothat they do not enter the detector in order to prevent damage to thedetector.

The ions entering the MS from ionisation of sample material (such as aplume of material generated by laser ablation) do not enter the MS allat the same time, but instead enter as a peak with a frequency thatfollows a probability distribution curve about a maximum frequency: frombaseline, at first a small number of ions enters the MS and aredetected, and then the frequency of ions increases to a maximum beforethe number decreases again and trails off to baseline. An event likelyto damage the detector can be identified because instead of a slowincrease in the frequency of ions at the leading edge of the peak, thereis a very quick increase in counts of ions hitting the detector.

The flow of ions hitting the detector of a TOF MS, a particular type ofdetector as discussed below, is not continual during the analysis of theions in a packet of ionised sample material. The TOF comprises a pulserwhich releases the ions periodically into the flight chamber of the TOFMS in pulsed groups. By releasing the ions all at the known same time,the time of flight mass determination is enabled. The time between thereleases of pulses of ions for time of flight mass determination isknown as an extraction or push of the TOF MS. The push is in the orderof microseconds. The signal from one or more packets of ions from thesampling and ionisation system therefore covers a number of pushes.

Accordingly, when the ion count reading jumps from the baseline to avery high count within one push (i.e. the first portion of the ions froma particular packet of ionised sample material) then it can be predictedthat the main body of ions resulting from ionisation of the packet ofsample material will be even greater, and so exceed the upper detectionlimit. It is at this point that an ion deflector can be operated toensure that the damaging bulk of the ions are directed away from thedetector (by being activated or deactivated, depending on thearrangement of the system, as discussed above).

Suitable ion deflectors based on quadrupoles are available in the art(e.g. from Colutron Research Corporation and Dreebit GmbH).

Laser Ablation-Based Sampling Systems of the Invention

The components of the laser ablation-based sampling systems can becombined as appropriate for the analytical task being undertaken.Exemplary embodiments are set out below.

In some embodiments, the laser ablation-based sampling system foranalysing a sample, such as a biological sample, comprises:

-   -   a sample stage;    -   a laser source;    -   a laser scanning system; and    -   focusing optics comprising an objective lens, wherein the        objective lens has a numerical aperture of at least 0.7, at        least 0.8 or at least 0.9, optionally further comprising an        immersion medium between the objective lens and the sample        stage.

In some embodiments, the laser ablation-based sampling system foranalysing a sample, such as a biological sample, comprises:

-   -   a sample stage;    -   a laser source comprising a femtosecond laser;    -   a laser scanning system; and    -   focusing optics comprising an objective lens, wherein the        objective lens has a numerical aperture of at least 0.7, at        least 0.8 or at least 0.9, optionally further comprising an        immersion medium between the objective lens and the sample        stage.

In some embodiments, the laser ablation-based sampling system foranalysing a sample, such as a biological sample, comprises:

-   -   a sample stage comprising a first face and a second face, the        first and second faces being opposed, and wherein the first face        is adapted to receive a sample;    -   a laser source;    -   a laser scanning system; and    -   focusing optics comprising an objective lens, the focusing        optics adapted to direct a beam of radiation from the laser        source towards the second face to a location on the sample        stage; and wherein the objective lens has a numerical aperture        of at least 0.7, at least 0.8 or at least 0.9, optionally        further comprising an immersion medium between the objective        lens and the sample stage.

In some embodiments, the laser ablation-based sampling system foranalysing a sample, such as a biological sample, comprises:

-   -   a sample stage comprising a first face and a second face, the        first and second faces being opposed, and wherein the first face        is adapted to receive a sample;    -   a laser source comprising a femtosecond laser;    -   a laser scanning system; and    -   focusing optics comprising an objective lens, the focusing        optics adapted to direct a beam of radiation from the laser        source towards the second face to a location on the sample        stage; and wherein the objective lens has a numerical aperture        of at least 0.7, at least 0.8 or at least 0.9, optionally        further comprising an immersion medium between the objective        lens and the sample stage.

In some embodiments, the laser ablation-based sampling system foranalysing a sample, such as a biological sample, comprises:

-   -   a sample stage comprising a first face and a second face, the        first and second faces being opposed, and wherein the first face        is adapted to receive a sample;    -   a laser source; and    -   focusing optics comprising an objective lens, the focusing        optics adapted to direct a beam of radiation from the laser        source towards the second face to a location on the sample        stage; and wherein the objective lens has a numerical aperture        of at least 0.7, at least 0.8 or at least 0.9, optionally        further comprising an immersion medium between the objective        lens and the sample stage.

In some embodiments, the laser ablation-based sampling system foranalysing a sample, such as a biological sample, comprises:

-   -   a sample stage comprising a first face and a second face, the        first and second faces being opposed, and wherein the first face        is adapted to receive a sample;    -   a laser source comprising a femtosecond laser; and    -   focusing optics comprising an objective lens, the focusing        optics adapted to direct a beam of radiation from the laser        source towards the second face to a location on the sample        stage; and wherein the objective lens has a numerical aperture        of at least 0.7, at least 0.8 or at least 0.9, optionally        further comprising an immersion medium between the objective        lens and the sample stage.

In some embodiments, the laser ablation-based sampling system foranalysing a sample, such as a biological sample, comprises:

-   -   a sample stage comprising a first face and a second face, the        first and second faces being opposed, and wherein the first face        is adapted to receive a sample;    -   a laser source;    -   a laser scanning system; and    -   focusing optics comprising an objective lens, optionally further        comprising an immersion medium between the objective lens and        the sample stage.

In some embodiments, the laser ablation-based sampling system foranalysing a sample, such as a biological sample, comprises:

-   -   a sample stage comprising a first face and a second face, the        first and second faces being opposed, and wherein the first face        is adapted to receive a sample;    -   a laser source comprising a femtosecond laser;    -   a laser scanning system; and    -   focusing optics comprising an objective lens, optionally further        comprising an immersion medium between the objective lens and        the sample stage.

In some embodiments, the laser ablation-based sampling system foranalysing a sample, such as a biological sample, comprises:

-   -   a sample stage comprising a first face and a second face, the        first and second faces being opposed, and wherein the first face        is adapted to receive a sample;    -   a laser source comprising a femtosecond laser; and    -   focusing optics comprising an objective lens, optionally further        comprising an immersion medium between the objective lens and        the sample stage.

Each of the laser ablation-based sampling systems set out above aresuitable for inclusion in an apparatus of the invention for analysing asample, comprising a mass detector (e.g. a TOF) mass detector, inparticular comprising an ICP ionisation system.

b. Sputtering Based Sampling and Ionising System

Sputtering based sampling systems and techniques provide alternativesurface analysis techniques to the laser ablation-based systems andtechniques described above. One such sputtering technique is SecondaryIon Mass Spectrometry (SIMS). SIMS involves bombarding a sample with afocused ion beam to sputter material from the sample. The sputteredmaterial comprises both ions and neutral atoms. In SIMS, the ions arethen transferred to a mass detector in a vacuum following capture by animmersion lens. The mass detector can be any of the mass detectorsystems described below. Similar sputtering can be achieved by directionof other charged particles at the sample, for instance electrons.

SIMS is a useful surface analysis technique for several reasons.Firstly, the technique is very sensitive to low concentrations ofanalyte material. Secondly, because diffraction effects of primary ionscan be neglected for most practical conditions, there is virtually nodiffraction limit in SIMS. Thus, SIMS has the potential to analysematerial on the scale of 10 to 30 nm.

However, the ionisation efficiency for the sputtered material is verylow and ionisation is also very dependent on the surface chemistry andspecific element being ionised, hence, the number of ions produced bySIMS is not always sufficient to provide a sufficient signal-to-noiseratio. For instance, an ionization efficiency is insufficient fordetection of single copies of antibodies labelled with MaxPar reagents.Since a single antibody labelled with MaxPar mass tag carries about 100atoms approximately 100 copies of antibodies may be required to generatea signal that is larger than a few ions at the detector in a traditionalSIMS workflow. Yet another deficiency of SIMS is due to spectralinterferences from molecules in the same mass channels and due toformations of compound ions such as oxides and other species involvingprimary tagging elements and abundant neutral atoms present inbiological samples. The compound ions dilute the signal of the elementalions and cause an overlap with mass channels of higher mass elements.Thus, the resolution of imaging using SIMS can be limited by lowsensitivity of detection due in part to low ionization efficiency.

The present invention overcomes the limitations of SIMS by providingimproved methods and apparatus for analysing a biological sample usinglaser-based Secondary Neutral Mass Spectrometry (SNMS).

The Laser-SNMS method and apparatus of the present invention involvebombarding a sample with a focused charged particle beam to sputtermaterial from the sample. A laser is used to post-ionise the neutralsputtered material. These ejected ions (including any detectable ionsfrom labelling atoms as discussed below) can be detected by a detectorsystem for instance a mass spectrometer (detectors are discussed in moredetail below). Since the majority of sputtered material is in theneutral state and SNMS ionises sputtered material so that it can beanalysed using mass detectors, SNMS provides a better quantitativeestimation of the surface than SIMS. Furthermore, as discussed above,one of the main challenges in improving the spatial resolution oftraditional IMS and IMC is ensuring that the amount of analyte in thematerial analysed provides a sufficient signal-to-noise ratio.Therefore, because SNMS makes use of both neutral and ionised sputteredmaterial, SNMS based IMC and IMS provide increased resolution comparedto SIMS based IMC and IMS. For instance, SNMS can enable single copydetection with antibodies tagged by MaxPar reagents. For instance, ifefficiency of postionization reaches 10% then 100 atoms per eachantibody would result in 10 ions produced per antibody and if these ionsare carried to the detector with good efficiency this would ensure aconfident detection of each copy of antibody.

A laser-SNMS system typically comprises three components. The firstcomponent is a source of charged particles for sputtering material fromthe sample for analysis (this source of charged particles is discussedin more detail below). The second component is a laser for post-ionisingthe sputtered material. The third component is a detector component thatdetects the ionised material, for instance a mass detector. Inlaser-SNMS, the laser and charged particle source are typically pulsed.In a related system, the first component is a source of chargedparticles for directed at a location on the sample (this source ofcharged particles is discussed in more detail below). The secondcomponent is a laser for causing ablation and optionally ionization ofthe locus at which has been preseeded with electrons by the chargedparticles. The third component is a detector component that detects theionised material, for instance a mass detector. In certain aspects, thespot size of the charged particles impinging the sample is smaller thanthe spot size of the laser. The charged particles may ablate sample atthe location, after which the laser may ionize sample near the samplesurface (e.g., as shown in FIG. 6). The charged particles may seedelectrons at the location of the sample, after which the laser mayablate and ionize sample that was seeded with electrons (e.g., as shownin FIG. 7). The laser may ionize the sample within picoseconds (e.g.,10-100 ps) of the charged particles impinging the sample (e.g., withinthe charge-ignition state). In certain aspects, the spot size of thecharged particles impinging the sample is smaller than the spot size ofthe laser, such as less than one half, less than one fifth, less thanone tenth, less than one twentieth, or less than one hundredth the sizeof the laser spot size. For example, the laser may have a spot size(impinging the sample) of less than 10 micrometres and/or more than 500nanometres, such as between 500 nanometers and 5 micrometers, between800 nm and 2 micrometer, or around 1 micrometer. The charged particlesmay provide a small spot size (e.g., in the range described herein) toallow ionization with minimal neutralization upon laser radiation, suchas with a spot size less than 200 nm, less than 100 nm, less than 50 nm,less than 30 nm, or less than 10 nm in diameter.

Accordingly, the invention provides an apparatus for analysing a sample,such as a biological sample, comprising:

-   -   a sample stage;    -   a source of charged particles and a charged particle column for        passing a beam of charged particles to a location on the sample        stage; and    -   a first laser source and first focusing optics configured to        direct a laser beam emitted by the first laser source towards        the sample stage.

The apparatus typically comprises a mass detector, such as a TOFdetector.

Accordingly, the invention provides a sampling and ionisation system foranalysing a sample, such as a biological sample, comprising:

-   -   a sample stage;    -   a source of charged particles and a charged particle column for        passing a beam of charged particles to a location on the sample        stage; and    -   a first laser source and first focusing optics configured to        direct a laser beam emitted by the first laser source towards        the sample stage.

The charged particle column of the present invention includesappropriate ion optics arranged to focus the charged particles in orderto pass the beam to a location on the sample stage. Such appropriate ionoptics can include a mass filter, lenses and apertures and deflectionplates in order to shape the primary ion beam, as described in moredetail below.

FIG. 6 is a schematic diagram of the arrangement of an exemplaryembodiment of the invention. The energy source 40 emits radiation (e.g.a laser beam, primary ion beam, or an electron beam, as discussedfurther below) which is passed towards a location 55 on the sample stage20 by optics 80 (e.g., light optics or ion optics, such as a chargedparticle column). The sample 30 is positioned on sample stage 20 suchthe charged particles passed towards location 55 sputter material 50from the sample 30. A first laser source 60 emits a laser beam andfocusing optics (not shown) direct the laser beam towards the samplestage to ionise the sputtered material 50, forming a plume of materialcomprising sample ions. The ions can then be transferred to a massdetector, for example a time of flight detector or magnetic sectordetector, or any other mass detector which is discussed in more detailbelow.

Accordingly, the invention provides an apparatus or sampling andionisation system for analysing a sample, such as a biological sample,wherein the first focusing optics is configured to synchronise a pulseof laser beam to ionise a plume of material sputtered by a pulse ofcharged particles (termed herein post-ionisation).

Accordingly, the invention provides an apparatus for analysing a sample,such as a biological sample, comprising:

-   -   a sample stage;    -   a source of charged particles and a charged particle column for        passing a beam of charged particles to a location on the sample        stage;    -   a first laser source and first focusing optics configured to        direct a laser beam emitted by the first laser source towards        the sample stage, wherein the first focusing optics is        configured to synchronise a pulse of laser beam to ionise a        plume of material sputtered by a pulse of charged particles from        the source of charged particles.

In particular, the first focussing optics direct and focus the laserbeam to a volume above the surface of the sample stage, such that whenmaterial is sputtered from a sample on the sample stage, the plume ofmaterial which is ejected from the sample passes into the volume towhich the radiation of the first laser source is focussed, such that thematerial can be ionized. This explanation applies to all apparatusdiscussed below in this section using the combination of features of asample stage, source of charged particles and first laser source andfirst focussing optics that ionise plume. It is merely not repeated eachtime below in the interests of brevity.

The apparatus typically comprises a mass detector, such as a TOFdetector.

Accordingly, the invention provides a sampling and ionisation system foranalysing a sample, such as a biological sample, comprising:

-   -   a sample stage;    -   a source of charged particles and a charged particle column for        passing a beam of charged particles to a location on the sample        stage; and    -   a first laser source and first focusing optics configured to        direct a laser beam emitted by the first laser source towards        the sample stage, wherein the first focusing optics is        configured to synchronise a pulse of laser beam to ionise a        plume of material sputtered by a pulse of charged particles from        the source of charged particles.

As explained above, the first focussing optics direct and focus thelaser beam to a volume above the location on the sample stage (morespecifically above the location on a sample on the sample stage), suchthat when material is sputtered from a sample on the sample stage, theplume of material which is ejected from the sample passes into thevolume to which the radiation of the first laser source is focussed,such that the material can be ionized. This explanation applies to allsystems discussed below in this section using the combination offeatures of a sample stage, source of charged particles and first lasersource and first focussing optics that ionise plume. It is merely notrepeated each time below in the interests of brevity.

FIG. 7 is a schematic diagram of the arrangement of a further exemplaryembodiment of the invention. FIG. 7 includes elements in common withFIG. 6 and these elements share the same reference numeral. However, inthe apparatus of the embodiment shown in FIG. 7, the laser beam fromfirst laser source 60 is directed towards the location 55 on the samplestage 20 at which the optics 80 (e.g., charged particle column) isdirected. For example, a pulse of the charged particles passed towardsthe location 55 on the sample forms an excited state with free electronsat the location 55 on the sample; this excited state is referred toherein as the ‘sample ignition state’. The laser beam from the firstlaser source illuminates the location 55 on the sample directly afterthe pulse of charged particles arrives at the location so that the firstlaser source illuminates the sample in the ignition state. Because thesample is in the sample ignition state, the sample readily converts thepulse of laser light from the laser beam into ablation and ionisationenergy; this state is referred to herein as the ‘sample energy pumpingstate’. This process forms a plume of material comprising sample ions.The skilled person would understand that the energy of the laser pulsein this embodiment is less than the ablation threshold of the specimen(such that areas of the same neighbouring that location to which thecharged particles were directed does not ablate in response to the pulseof laser light that causes the location to enter ‘sample energy pumpingstate’, as the laser spot on the sample can be larger than the diameterof the location to which charged particles were directed).

Accordingly, the invention provides an apparatus for analysing a sample,such as a biological sample, wherein the first focusing optics isconfigured to synchronise a pulse of laser beam to arrive at thelocation on the sample stage directly after a pulse of chargedparticles. This configuration of the present invention can overcome therelatively slow sputtering of particles by the charged particle beamalone and hence provide a quicker method of analysing a biologicalsample.

Accordingly, the invention provides an apparatus for analysing a sample,such as a biological sample, comprising:

-   -   a sample stage;    -   a source of charged particles and a charged particle column for        passing a beam of charged particles to a location on the sample        stage; and    -   a first laser source and first focusing optics configured to        direct a laser beam emitted by the first laser source towards        the sample stage, wherein the first focusing optics is        configured to synchronise a pulse of the laser beam to arrive at        the location on the sample stage directly after a pulse of        charged particles.

In particular, the first focussing optics direct and focus the laserbeam/laser radiation to the same location on a sample on the samplestage as previously targeted by the charged particles. This explanationapplies to all apparatus discussed below in this section using thecombination of features of a sample stage, source of charged particlesand first laser source and first focussing optics that causes a pulse ofradiation from the first laser source to illuminate the same location aspreviously targeted by the charged particles. It is merely not repeatedeach time below in the interests of brevity.

The apparatus typically comprises a mass detector, such as a TOFdetector.

Accordingly, the invention provides a sampling and ionisation system foranalysing a sample, such as a biological sample, comprising:

-   -   a sample stage;    -   a source of charged particles and a charged particle column for        passing a beam of charged particles to a location on the sample        stage; and    -   a first laser source and first focusing optics configured to        direct a laser beam emitted by the first laser source towards        the sample stage, wherein the first focusing optics is        configured to synchronise a pulse of the laser beam to arrive at        the location on the sample stage directly after a pulse of        charged particles.

In particular, the first focussing optics direct and focus the laserbeam/laser radiation to the same location on a sample on the samplestage as previously targeted by the charged particles. This explanationapplies to all systems discussed below in this section using thecombination of features of a sample stage, source of charged particlesand first laser source and first focussing optics that causes a pulse ofradiation from the first laser source to illuminate the same location aspreviously targeted by the charged particles. It is merely not repeatedeach time below in the interests of brevity. FIG. 8 is a schematicdiagram of the arrangement of a further exemplary embodiment of theinvention. FIG. 8 includes elements in common with FIGS. 6 and 7 andthese elements share the same reference numeral. In this embodiment, thesample stage 20 is transparent (as is any sample carrier on which thesample is positioned). The skilled person would understand thattransparent sample stages or sample stages comprising a cut-out portioncould be used with any of the embodiments of the sputtering basedsampling and ionising systems described herein. Transparent samplestages are described in more detail herein.

In the embodiment shown in FIG. 8, the source of charged particles andcharged particle column, and first laser source and the first focusingoptics are configured such that the beam of charged particles and thelaser beam are directed towards the opposite sides of the sample stage.In the embodiment shown in FIG. 8, the charged particles are passedtowards the location 55 on the sample and the laser beam is alsodirected towards the location 55 on the sample. In the same way as theembodiment shown in FIG. 7, in the embodiment of FIG. 8, a pulsed beamof charged particles provides a sample ignition state and a pulsed laserbeam provides an energy pumping state to result in a plume of materialcomprising sample ions.

FIG. 9 is a schematic diagram of the arrangement of a further exemplaryembodiment of the invention. FIG. 9 includes elements in common withFIGS. 6 to 8 and these elements share the same reference numeral.Similarly to the embodiment shown in FIG. 8, in this embodiment of theinvention, the source of charged particles and charged particle column,and first laser source and the first focusing optics are configured suchthat the beam of charged particles and the laser beam are directedtowards the opposite sides of the sample stage. The sample 30 ispositioned on sample stage 20 such the charged particles passed towardslocation 55 sputter material 50 from the sample 30. A first laser source60 emits a laser beam and focusing optics (not shown) direct the laserbeam towards the sample stage to ionise the sputtered material 50,forming a plume of material comprising sample ions. The ions can then betransferred to a mass detector, for example a time of flight detector,or any other mass detector which is discussed in more detail below.

Of note, in any of the above embodiments, the charged particle beam maybe scanned across the sample to analyse an arbitrary region of interest,such as an organelle, as discussed earlier.

Accordingly, invention provides for an apparatus wherein the source ofcharged particles and charged particle column, and the first lasersource and first focusing optics, are configured such that the beam ofcharged particles and laser beam are directed towards the same side ofthe sample stage. Examples of this type of apparatus are shown in FIGS.6 and 7.

Accordingly, the invention provides an apparatus for analysing a sample,such as a biological sample, comprising:

-   -   a sample stage;    -   a source of charged particles and a charged particle column for        passing a beam of charged particles to a location on the sample        stage; and    -   a first laser source and first focusing optics configured to        direct a laser beam emitted by the first laser source towards        the sample stage, wherein the first focusing optics is        configured to synchronise a pulse of the laser beam to arrive at        the location on the sample stage directly after a pulse of        charged particles; and    -   wherein the source of charged particles and charged particle        column, and the first laser source and first focusing optics,        are configured such that the beam of charged particles and laser        beam are directed towards the same side of the sample stage.

The apparatus typically comprises a mass detector, such as a TOFdetector.

Accordingly, the invention provides a sampling and ionisation system foranalysing a sample, such as a biological sample, comprising:

-   -   a sample stage;    -   a source of charged particles and a charged particle column for        passing a beam of charged particles to a location on the sample        stage; and    -   a first laser source and first focusing optics configured to        direct a laser beam emitted by the first laser source towards        the sample stage, wherein the first focusing optics is        configured to synchronise a pulse of the laser beam to arrive at        the location on the sample stage directly after a pulse of        charged particles; and    -   wherein the source of charged particles and charged particle        column, and the first laser source and first focusing optics,        are configured such that the beam of charged particles and laser        beam are directed towards the same side of the sample stage.

For instance, the invention provides an apparatus for analysing asample, such as a biological sample, comprising:

-   -   a sample stage comprising a first face and a second face, the        first and second faces being opposed, and wherein the first face        is adapted to receive a sample;    -   a source of charged particles and a charged particle column for        passing a beam of charged particles to a location on the first        face of the sample stage; and    -   a first laser source and first focusing optics configured to        direct a laser beam emitted by the first laser source towards        the first face of the sample stage, wherein the first focusing        optics is configured to synchronise a pulse of the laser beam to        arrive at the location on the sample stage directly after a        pulse of charged particles

The apparatus typically comprises a mass detector, such as a TOFdetector.

For instance, the invention provides a sampling and ionisation systemfor analysing a sample, such as a biological sample, comprising:

-   -   a sample stage comprising a first face and a second face, the        first and second faces being opposed, and wherein the first face        is adapted to receive a sample;    -   a source of charged particles and a charged particle column for        passing a beam of charged particles to a location on the first        face of the sample stage; and    -   a first laser source and first focusing optics configured to        direct a laser beam emitted by the first laser source towards        the first face of the sample stage, wherein the first focusing        optics is configured to synchronise a pulse of the laser beam to        arrive at the location on the sample stage directly after a        pulse of charged particles.

Accordingly, the invention provides an apparatus for analysing a sample,such as a biological sample, comprising:

-   -   a sample stage;    -   a source of charged particles and a charged particle column for        passing a beam of charged particles to a location on the sample        stage; and    -   a first laser source and first focusing optics configured to        direct a laser beam emitted by the first laser source towards        the sample stage, wherein the first focusing optics is        configured to synchronise a pulse of laser beam to ionise a        plume of material sputtered by a pulse of charged particles from        the source of charged particles; and    -   wherein the source of charged particles and charged particle        column, and the first laser source and first focusing optics,        are configured such that the beam of charged particles and laser        beam are directed towards the same side of the sample stage.

The apparatus typically comprises a mass detector, such as a TOFdetector.

Accordingly, the invention provides a sampling and ionisation system foranalysing a sample, such as a biological sample, comprising:

-   -   a sample stage;    -   a source of charged particles and a charged particle column for        passing a beam of charged particles to a location on the sample        stage; and    -   a first laser source and first focusing optics configured to        direct a laser beam emitted by the first laser source towards        the sample stage, wherein the first focusing optics is        configured to synchronise a pulse of laser beam to ionise a        plume of material sputtered by a pulse of charged particles from        the source of charged particles; and    -   wherein the source of charged particles and charged particle        column, and the first laser source and first focusing optics,        are configured such that the beam of charged particles and laser        beam are directed towards the same side of the sample stage.

For instance, the invention provides an apparatus for analysing asample, such as a biological sample, comprising:

-   -   a sample stage comprising a first face and a second face, the        first and second faces being opposed, and wherein the first face        is adapted to receive a sample;    -   a source of charged particles and a charged particle column for        passing a beam of charged particles to a location on the first        face of the sample stage; and    -   a first laser source and first focusing optics configured to        direct a laser beam emitted by the first laser source towards        the first face of the sample stage, wherein the first focusing        optics is configured to synchronise a pulse of laser beam to        ionise a plume of material sputtered by from the sample by a        pulse of charged particles from the source of charged particles.

The apparatus typically comprises a mass detector, such as a TOFdetector.

For instance, the invention provides a sampling and ionisation systemfor analysing a sample, such as a biological sample, comprising:

-   -   a sample stage comprising a first face and a second face, the        first and second faces being opposed, and wherein the first face        is adapted to receive a sample;    -   a source of charged particles and a charged particle column for        passing a beam of charged particles to a location on the first        face of the sample stage; and    -   a first laser source and first focusing optics configured to        direct a laser beam emitted by the first laser source towards        the first face of the sample stage, wherein the first focusing        optics is configured to synchronise a pulse of laser beam to        ionise a plume of material sputtered by from the sample by a        pulse of charged particles from the source of charged particles.

In addition, the invention provides an apparatus wherein the source ofcharged particles and charged particle column, and the first lasersource and first focusing optics, are configured such that the beam ofcharged particles and laser beam are directed towards opposite sides ofthe sample stage. Examples of this type of apparatus are shown in FIGS.8 and 9. An advantage of these types of set-up is that they minimise themechanical complications of combining the charged particle column andthe focusing optics. In certain aspects, the charged particle may bedirected toward the sample through the support or substrate that is atleast partially transparent to the charged particles, and ablate thesample or seed electrons in the sample. In such aspects, the sample maybe thin, such as less than 200 nm, less than 100 nm, less than 50 nm, orless than 30 nm.

Accordingly, the invention provides an apparatus for analysing a sample,such as a biological sample, comprising:

-   -   a sample stage;    -   a source of charged particles and a charged particle column for        passing a beam of charged particles to a location on the sample        stage; and    -   a first laser source and first focusing optics configured to        direct a laser beam emitted by the first laser source towards        the sample stage, wherein the first focusing optics is        configured to synchronise a pulse of the laser beam to arrive at        the location on the sample stage directly after a pulse of        charged particles; and    -   wherein the source of charged particles and charged particle        column, and the first laser source and first focusing optics,        are configured such that the beam of charged particles and laser        beam are directed towards opposite sides of the sample stage.

The apparatus typically comprises a mass detector, such as a TOFdetector.

Accordingly, the invention provides a sampling and ionisation system foranalysing a sample, such as a biological sample, comprising:

-   -   a sample stage;    -   a source of charged particles and a charged particle column for        passing a beam of charged particles to a location on the sample        stage; and    -   a first laser source and first focusing optics configured to        direct a laser beam emitted by the first laser source towards        the sample stage, wherein the first focusing optics is        configured to synchronise a pulse of the laser beam to arrive at        the location on the sample stage directly after a pulse of        charged particles; and    -   wherein the source of charged particles and charged particle        column, and the first laser source and first focusing optics,        are configured such that the beam of charged particles and laser        beam are directed towards opposite sides of the sample stage.

For instance, the invention provides an apparatus for analysing asample, such as a biological sample, comprising:

-   -   a sample stage comprising a first face and a second face, the        first and second faces being opposed, and wherein the first face        is adapted to receive a sample;    -   a source of charged particles and a charged particle column for        passing a beam of charged particles to a location on the first        face of the sample stage; and    -   a first laser source and first focusing optics configured to        direct a laser beam emitted by the first laser source towards        the second face of the sample stage, wherein the first focusing        optics is configured to synchronise a pulse of the laser beam to        arrive at the location on the sample stage directly after a        pulse of charged particles

The apparatus typically comprises a mass detector, such as a TOFdetector.

For instance, the invention provides a sampling and ionisation systemfor analysing a sample, such as a biological sample, comprising:

-   -   a sample stage comprising a first face and a second face, the        first and second faces being opposed, and wherein the first face        is adapted to receive a sample;    -   a source of charged particles and a charged particle column for        passing a beam of charged particles to a location on the first        face of the sample stage; and    -   a first laser source and first focusing optics configured to        direct a laser beam emitted by the first laser source towards        the second face of the sample stage, wherein the first focusing        optics is configured to synchronise a pulse of the laser beam to        arrive at the location on the sample stage directly after a        pulse of charged particles.

As explained elsewhere herein, and reiterated here for completeness,when the laser beam is recited through the sample stage to reach asample on the first face, the stage should be transparent to the laserradiation (as should the sample carrier for the sample), or the samplestage should include a void through which laser radiation can pass toreach the sample (through the sample carrier)

The skilled person would also understand that the configurations ofFIGS. 6 to 9 can be combined in various ways. FIG. 10 shows a furtherexemplary embodiment of the invention which is a combination of theconfigurations of FIGS. 6 to 9, again elements in common with FIGS. 6 to9 are labelled with the same reference numeral.

The embodiment shown in FIG. 10 includes a second laser source 61 andsecond focusing optics (not shown). Similarly to the first laser source60 and second focusing optics of FIG. 8, the second laser source 61 andsecond focusing optics of this embodiment are configured to direct asecond laser beam towards location 55 on the sample stage. In the sameway as the embodiments shown in FIGS. 7 and 8, the pulsed beam ofcharged particles provides a sample ignition state and a pulsed laserbeam from the second laser source 61 provides an energy pumping state toresult in a plume of material comprising sample ions. In this embodimentof the invention, the first laser source 60 and first focusing opticsare configured to ionise a plume of material sputtered by the pulse ofcharged particles. Therefore, the first laser source 60 will ionise anyneutral material sputtered from the surface not ionised by the energypumping state. Hence, since both the first and second laser source areconfigured to ionise a plume of material to produce sample ions thepresent invention provides an apparatus which further improvesionisation probability and so increases signal-to-noise ratio. Thus, theinvention provides an apparatus that analyses a sample with increasedresolution.

Accordingly, the present invention provides for an apparatus asdescribed above in this section comprising a second laser source andsecond focusing optics, wherein the second focusing optics areconfigured to synchronise a pulse of laser light from the second lasersource to ionise a plume of material sputtered by a pulse of chargedparticles. In certain aspects, the first and second laser sources cancomprise the same laser, wherein the control of the laser and/or opticsallow for the two-step laser radiation described herein.

Accordingly, the invention provides an apparatus for analysing a sample,such as a biological sample, comprising:

-   -   a sample stage;    -   a source of charged particles and a charged particle column for        passing a beam of charged particles to a location on the sample        stage;    -   a first laser source and first focusing optics configured to        direct a laser beam emitted by the first laser source towards        the sample stage, wherein the first focusing optics is        configured to synchronise a pulse of the laser beam to arrive at        the location on the sample stage directly after a pulse of        charged particles; and    -   a second laser source and second focusing optics, wherein the        second focusing optics are configured to synchronise a pulse of        the laser beam from the second laser source to ionise a plume of        sample material from the location on the sample, generated by        the pulse of charged particles and the pulse of the laser beam        from the first laser source.

The apparatus typically comprises a mass detector, such as a TOFdetector.

Accordingly, the invention provides a sampling and ionisation system foranalysing a sample, such as a biological sample, comprising:

-   -   a sample stage;    -   a source of charged particles and a charged particle column for        passing a beam of charged particles to a location on the sample        stage;    -   a first laser source and first focusing optics configured to        direct a laser beam emitted by the first laser source towards        the sample stage, wherein the first focusing optics is        configured to synchronise a pulse of laser beam to ionise a        plume of material sputtered by a pulse of charged particles from        the source of charged particles; and    -   a second laser source and second focusing optics, wherein the        second focusing optics are configured to synchronise a pulse of        the laser beam from the second laser source to ionise a plume of        sample material from the location, generated by the pulse of        charged particles and the pulse of the laser beam from the first        laser source.

Accordingly, the present invention provides for an apparatus wherein thefirst laser source and first focusing optics and second laser source andsecond focusing optics, are configured such that the laser beam from thefirst laser source and laser beam from the second laser source aredirected towards are directed towards the same side of the sample stage.

Accordingly, the invention provides an apparatus for analysing a sample,such as a biological sample, comprising:

-   -   a sample stage;    -   a source of charged particles and a charged particle column for        passing a beam of charged particles to a location on the sample        stage;    -   a first laser source and first focusing optics configured to        direct a laser beam emitted by the first laser source towards        the sample stage, wherein the first focusing optics is        configured to synchronise a pulse of the laser beam to arrive at        the location on the sample stage directly after a pulse of        charged particles; and    -   a second laser source and second focusing optics, wherein the        second focusing optics are configured to synchronise a pulse of        the laser beam from the second laser source to ionise a plume of        sample material from the location on the sample, generated by        the pulse of charged particles and the pulse of the laser beam        from the first laser source, wherein the source of charged        particles and charged particle column, the first laser source        and first focusing optics, and the second laser source and        second focusing optics are configured such that the beam of        charged particles, laser beam from the first laser source and        laser beam from the second laser source are directed towards the        same side of the sample stage.

The apparatus typically comprises a mass detector, such as a TOFdetector.

Accordingly, the invention provides a sampling and ionisation system foranalysing a sample, such as a biological sample, comprising:

-   -   a sample stage;    -   a source of charged particles and a charged particle column for        passing a beam of charged particles to a location on the sample        stage;    -   a first laser source and first focusing optics configured to        direct a laser beam emitted by the first laser source towards        the sample stage, wherein the first focusing optics is        configured to synchronise a pulse of the laser beam to arrive at        the location on the sample stage directly after a pulse of        charged particles; and    -   a second laser source and second focusing optics, wherein the        second focusing optics are configured to synchronise a pulse of        the laser beam from the second laser source to ionise a plume of        sample material from the location on the sample, generated by        the pulse of charged particles and the pulse of the laser beam        from the first laser source, wherein the source of charged        particles and charged particle column, the first laser source        and first focusing optics, and the second laser source and        second focusing optics are configured such that the beam of        charged particles, laser beam from the first laser source and        laser beam from the second laser source are directed towards the        same side of the sample stage.

For instance, the invention provides an apparatus for analysing asample, such as a biological sample, comprising:

-   -   a sample stage comprising a first face and a second face, the        first and second faces being opposed, and wherein the first face        is adapted to receive a sample;    -   a source of charged particles and a charged particle column for        passing a beam of charged particles to a location on the first        face of the sample stage;    -   a first laser source and first focusing optics configured to        direct a laser beam emitted by the first laser source towards        the first face of the sample stage, wherein the first focusing        optics is configured to synchronise a pulse of the laser beam to        arrive at the location on the first face of the sample stage        directly after a pulse of charged particles; and    -   a second laser source and second focusing optics configured to        direct a laser beam emitted by the second laser source towards        the first face of the sample stage, wherein the second focusing        optics are configured to synchronise a pulse of the laser beam        from the second laser source to ionise a plume of sample        material from the location on the sample, generated by the pulse        of charged particles and the pulse of the laser beam from the        first laser source.

The apparatus typically comprises a mass detector, such as a TOFdetector.

Accordingly, the invention provides a sampling and ionisation system foranalysing a sample, such as a biological sample, comprising:

-   -   a sample stage comprising a first face and a second face, the        first and second faces being opposed, and wherein the first face        is adapted to receive a sample;    -   a source of charged particles and a charged particle column for        passing a beam of charged particles to a location on the first        face of the sample stage;    -   a first laser source and first focusing optics configured to        direct a laser beam emitted by the first laser source towards        the first face of the sample stage, wherein the first focusing        optics is configured to synchronise a pulse of the laser beam to        arrive at the location on the first face of the sample stage        directly after a pulse of charged particles; and    -   a second laser source and second focusing optics configured to        direct a laser beam emitted by the second laser source towards        the first face of the sample stage, wherein the second focusing        optics are configured to synchronise a pulse of the laser beam        from the second laser source to ionise a plume of sample        material from the location on the sample, generated by the pulse        of charged particles and the pulse of the laser beam from the        first laser source.

Accordingly, the invention provides an apparatus wherein the first lasersource and first focusing optics and second laser source and secondfocusing optics, are configured such that the laser beam from the firstlaser source and laser beam from the second laser source are directedtowards opposite sides of the sample stage.

Accordingly, the invention provides an apparatus for analysing a sample,such as a biological sample, comprising:

-   -   a sample stage;    -   a source of charged particles and a charged particle column for        passing a beam of charged particles to a location on the sample        stage;    -   a first laser source and first focusing optics configured to        direct a laser beam emitted by the first laser source towards        the sample stage, wherein the first focusing optics is        configured to synchronise a pulse of the laser beam to arrive at        the location on the sample stage directly after a pulse of        charged particles; and    -   a second laser source and second focusing optics, wherein the        second focusing optics are configured to synchronise a pulse of        the laser beam from the second laser source to ionise a plume of        sample material from the location on the sample, generated by        the pulse of charged particles and the pulse of the laser beam        from the first laser source, wherein the source of charged        particles and charged particle column, and the second laser        source and second focusing optics are configured such that the        beam of charged particles and laser beam from the second laser        source are directed towards the same side of the sample stage;        and the first laser source and first focusing optics are        configured such that the laser beam from the first laser source        is directed towards the opposite side of the sample stage.

The apparatus typically comprises a mass detector, such as a TOFdetector.

Accordingly, the invention provides a sampling and ionisation system foranalysing a sample, such as a biological sample, comprising:

-   -   a sample stage;    -   a source of charged particles and a charged particle column for        passing a beam of charged particles to a location on the sample        stage;    -   a first laser source and first focusing optics configured to        direct a laser beam emitted by the first laser source towards        the sample stage, wherein the first focusing optics is        configured to synchronise a pulse of the laser beam to arrive at        the location on the sample stage directly after a pulse of        charged particles; and    -   a second laser source and second focusing optics, wherein the        second focusing optics are configured to synchronise a pulse of        the laser beam from the second laser source to ionise a plume of        sample material from the location on the sample, generated by        the pulse of charged particles and the pulse of the laser beam        from the first laser source, wherein the source of charged        particles and charged particle column, and the second laser        source and second focusing optics are configured such that the        beam of charged particles and laser beam from the second laser        source are directed towards the same side of the sample stage;        and the first laser source and first focusing optics are        configured such that the laser beam from the first laser source        is directed towards the opposite side of the sample stage.

For instance, the invention provides an apparatus for analysing asample, such as a biological sample, comprising:

-   -   a sample stage comprising a first face and a second face, the        first and second faces being opposed, and wherein the first face        is adapted to receive a sample;    -   a source of charged particles and a charged particle column for        passing a beam of charged particles to a location on the first        face of the sample stage;    -   a first laser source and first focusing optics configured to        direct a laser beam emitted by the first laser source towards        the second face of the sample stage, wherein the first focusing        optics is configured to synchronise a pulse of the laser beam to        arrive at the location on the first face of the sample stage        directly after a pulse of charged particles; and    -   a second laser source and second focusing optics configured to        direct a laser beam emitted by the second laser source towards        the first face of the sample stage, wherein the second focusing        optics are configured to synchronise a pulse of the laser beam        from the second laser source to ionise a plume of sample        material from the location,    -   generated by the pulse of charged particles and the pulse of the        laser beam from the first laser source.

The apparatus typically comprises a mass detector, such as a TOFdetector.

Accordingly, the invention provides a sampling and ionisation system foranalysing a sample, such as a biological sample, comprising:

-   -   a sample stage comprising a first face and a second face, the        first and second faces being opposed, and wherein the first face        is adapted to receive a sample;    -   a source of charged particles and a charged particle column for        passing a beam of charged particles to a location on the first        face of the sample stage;    -   a first laser source and first focusing optics configured to        direct a laser beam emitted by the first laser source towards        the second face of the sample stage, wherein the first focusing        optics is configured to synchronise a pulse of the laser beam to        arrive at the location on the first face of the sample stage        directly after a pulse of charged particles; and    -   a second laser source and second focusing optics configured to        direct a laser beam emitted by the second laser source towards        the first face of the sample stage, wherein the second focusing        optics are configured to synchronise a pulse of the laser beam        from the second laser source to ionise a plume of sample        material from the location,    -   generated by the pulse of charged particles and the pulse of the        laser beam from the first laser source.

The invention also provides methods of analysing a biological sampleusing an apparatus as described in this section. For instance, theinvention provides a method comprising:

-   -   passing a beam of charged particles towards a location on the        sample; illuminating the sample with a first pulse of laser beam        to produce a plume of material comprising sample ions; and    -   detecting said sample ions by mass spectrometry.

In some instances, the method comprises passing the beam of chargedparticles towards a location on the sample to sputter material from thesample; and wherein the method comprises illuminating the sputteredsample material with the first pulse of laser beam and ionising thesputtered material to produce sample ions. That is to say, the methodcomprises passing the beam of charged particles towards a location onthe sample to sputter material from the sample; and wherein the methodcomprises illuminating the sputtered sample material with the firstpulse of laser beam thereby ionising the sputtered material to producesample ions. Sometimes, passing the beam of charged particles towards alocation on the sample produces a sample ignition state; and whereinilluminating the sample at the location produces a sample energy pumpingstate at the location on the sample.

In some of the methods, the beam of charged particles is passed towardsa location on the sample from one side of the sample, and wherein thefirst pulse of laser beam illuminates the sample from the same side ofthe sample. In some embodiments the beam of charged particles is passedtowards a location on the sample from one side of the sample, andwherein the first pulse of laser beam illuminates the sample from theopposite side of the sample. Sometimes, passing the beam of chargedparticles towards a location on the sample further comprises sputteringmaterial from the sample; and wherein the method comprises illuminatingthe sputtered sample material with a second pulse of laser beam andionising the sputtered material. The first and second pulses of laserbeam illuminate the sample from the same side of the sample or the firstand second pulses of laser beam illuminate the sample from oppositesides of the sample.

As the skilled person will appreciate, the laser ionisation of thesputtered material can be accomplished by various ionisation mechanismsincluding single photon ionisation, resonant and non-resonantmultiphoton ionisation, avalanche ionisation and cold avalancheionisation. Furthermore, different ionisation mechanisms can operatesimultaneously, such as multiphoton ionisation and avalanche ionisation.

Single photon ionisation (SPI) is the process by which the absorption ofone photon is sufficient to overcome the ionisation potential sputteredmaterial. However, ionisation by this means requires very high energyultraviolet or vacuum ultraviolet lasers, for example excimer lasers, orcomplex systems employing non-linear optical processes in gases.Accordingly, the present invention provides methods and apparatuswherein the first and/or second laser source is a high energyultraviolet or vacuum ultraviolet laser.

Multiphoton ionisation (MPI) involves the absorption of more than onephoton in order to overcome the ionisation potential, this absorptioncan be in non-resonant or resonant steps. Multiphoton ionisationrequires short and intense laser pulses. Suitable lasers for resonantsystems include pulsed Nd:YAG laser pumping two dye lasers, suitablelasers for non-resonant laser systems include a high-power excimer orNd:YAg laser. Accordingly, the present invention provides methods andapparatus wherein the first and/or second laser source is a Nd:YAG laserpumping two dye lasers, a high-power excimer or Nd:YAG laser.

Avalanche ionisation (Al) is the process by which electrons collide withand ionise sputtered material, resulting in additional electrons whichaccelerate and collide with other sputtered material, thereby creating achain reaction. In the present invention, the initial ‘seed’ electronsmay be the result of any such ionisation mechanism that results from theapplication of a laser pulse, for example multiphoton ionisation orsingle photon ionisation. Some models imply that avalanche ionisation isunimportant when the laser pulse is shorter than 100 fs. However,various techniques have been demonstrated where avalanche ionisationpersists at laser pulses shorter than 100 fs. The present inventionprovides methods utilising these techniques in order to ionise sputteredmaterial by means of avalanche ionisation using laser pulses shorterthan 100 fs.

In the first technique, the electric field imposed by an intenseionising laser on the sample is used to reduce the effective energythreshold at which collisional ionisation occurs and so thus allowcollisional ionisation to drive avalanche ionisation even at short pulselengths (less than 100 fs). This is a cold avalanche ionisationmechanism. Suitable lasers may be, for example a Ti:sapphire laser (800nm, 40-45 fs pulse length, energy 75 nJ, e.g. Octavius Ti:SapphireLasers, available from Thorlabs) and it has been shown that avalancheionisation of fused silica can be achieved with 800 nm laser pulses aslow as 40 fs from a regenerative amplifier (Rajeev, Gertsvolf et al.2009, PRL 201). Accordingly, the present invention provides methods andapparatus wherein the first and/or second laser source is a Ti:sapphirelaser for ionising the sputtered material by avalanche ionisation. Incertain aspects, the mechanism of ignition (e.g., electron seedingseeding) followed by plasma development (e.g., avalanche ionisation) maybe similar for dielectrics and for biological samples. In the case ofbiological samples, the sample format may be in an epoxy resin which isa dielectric, though different than Silicon Oxide.

In the second technique, free carriers are injected into the sample toencourage exciton-seeded multiphoton ionisation in combination withavalanche ionisation. Free carriers can be injected into a dielectricfrom extreme ultraviolet sources created by high-harmonic or attosecondpulse generation and the sample can be ionised using a laser pulse asshort as 45 fs (800 nm laser), (Grojo, Gertsvolf et al. 2010, PR 81).Accordingly, the present invention provides methods and apparatuswherein the first and/or second laser source is an extreme ultravioletsource.

Accordingly, the present invention provides methods and apparatuswherein the first and/or second laser is configured to ionise sputteredmaterial by avalanche ionisation.

In this way, the present invention further addresses the two mainchallenges in enhancing the resolution of IMC to sub micrometer scales.Firstly, because laser pulses shorter than 100 fs can be used to effectavalanche ionisation, the risk of the area around the laser spot sizebeing damaged due to heating effects is reduced. In this way, thepresent invention maintains the spot area to a size of around 200 nm orless. Secondly, the present invention improves the likelihood ofmultiphoton and avalanche ionisation and therefore improves overallionisation rates. In this way, the overall amount of analyte in theablated material provides a sufficient signal-to-noise ratio.

Components of Sputtering Based Sampling and Ionising System

Source of Charged Particles

As discussed above, in the present invention, a source of chargedparticles and a charged particle column may be used to pass a beam ofcharged particles to a location on the sample.

Primary Ion Beam:

Typically, the source of charged particles used in secondary neutralmass spectrometry is a primary ion beam source. The primary ions can beany suitable ion for generating sputtering from the sample to beanalysed. Examples of primary ion sources are: the Duoplasmatron whichgenerates oxygen (¹⁶O⁻, ¹⁶O₂ ⁺, ¹⁶O₂ ⁻), argon (⁴⁰Ar⁺), xenon (Xe⁺), SF₅⁺, or C₆₀ ⁺ primary ions; a surface ionisation source which generates¹³³Cs⁺ primary ions; and liquid metal ion guns (LMIG) which generate Ga⁺primary ions. Other primary ions include cluster ions such as Au_(n) ⁺(n=1-5), Bi_(n) ^(q+) (n=1-7, q=1 and 12), C₆₀ ^(q+) probes (q=1-3) andlarge Ar clusters (Muramoto, Brison, & Castner, 2012).

The choice of ion source depends on the type of ion bombardment beingdeployed (i.e. static or dynamic) and the sample to be analysed. Staticinvolves using a low primary ion beam current (1 nA/cm²), usually apulsed ion beam. Because of the low current, each ion strikes a newsection of the sample surface, removing only a monolayer of particles (2nm). Hence, static is suitable for imaging and surface analysis (Gamble& Anderton, 2016). Dynamic involves using a high primary ion beamcurrent (10 mA/cm²), usually a continuous primary ion beam, whichresults in the fast removal of surface particles. As a result, ispossible to use dynamic for depth profiling. Furthermore, since morematerial is removed from the sample surface, dynamic SIMS gives a betterdetection limit than static. Dynamic typically produces high imageresolution (less than 100 nm) (Vickerman & Briggs, 2013).

Oxygen primary ions enhance ionisation of electropositive elements(Malherbe, Penen, Isaure, & Frank, 2016) and are used in thecommercially available Cameca IMS 1280-HR, whereas caesium primary ionsare used to investigate electronegative elements (Kiss, 2012) and areused in the commercially available Cameca NanoSIMS 50.

For rapid analysis of a sample a high frequency of sputtering is needed,for example more than 200 Hz (i.e. more than 200 packets of ionsdirected at the sample per second). Commonly, the frequency of primaryion pulse generation by the primary ion source is at least 400 Hz, suchas at least 500 Hz, or at least 1 kHz. For instance, the frequency ofion pulses in some embodiments is at least 10 kHz, at least 100 kHz, atleast 1 MHz, or at least 10 MHz. For instance, the frequency of ionpulses is within the range 400-100 MHz, within the range 1 kHz-100 MHz,within the range 10 kHz-100 MHz, within the range 100 kHz-100 MHz orwithin the range 1 MHz-100 MHz.

Accordingly, the present invention provides an apparatus wherein thesource of charged particles is a primary ion beam.

Electron Beam

Alternatively, in some embodiments of the present invention, the sourceof charged particles is an electron beam. Electron beams with the energyof 2 kV to 30 kV may be particularly suitable to interrogate a specimenwith a thickness of 30 nm.

A high intensity pulsed electron beam is used to causeablation/sputtering. When the pulse of the electron current isinsufficient for ablation, its effect can be used just as an ignitionevent as described above, followed by energy pumping by the laser pulseset at the brightness level below the level of ablation of nativematerial but above the level of energy pumping required for ablation ofan already activated material. In the energy activated mode ofablation/sputtering, the electron energy can be lowered as the electronsonly serve to inject charge carriers into otherwise insulating material.

In certain aspects, the electron beam may be focused at a smaller spot(at a higher resolution) than the laser beam. A laser of lowerresolution may only ablate and/or ignite sample at the spot radiated bythe electron beam, even when it impinges sample beyond the electron beamspot. For example, the electron beam may be focused to a spot of 200 nm,100 nm, 50 nm, 30 nm, or 10 nm or less and the laser may be focussed toa spot of 200 nm, 300 nm, 500 nm, 800 nm, 1 um, 2 um or more thatoverlaps with the electron beam spot or is focused on an ablation plumerelease by the electron beam.

For rapid analysis of a sample a high frequency of sputtering is needed,for example more than 200 Hz (i.e. more than 200 packets of electronsdirected at the sample per second). Commonly, the frequency of electronpulse generation by the electron source is at least 400 Hz, such as atleast 500 Hz, or at least 1 kHz. For instance, the frequency of electronpulses in some embodiments is at least 10 kHz, at least 100 kHz, atleast 1 MHz, or at least 10 MHz. For instance, the frequency of electronpulses is within the range 400-100 MHz, within the range 1 kHz-100 MHz,within the range 10 kHz-100 MHz, within the range 100 kHz-100 MHz orwithin the range 1 MHz-100 MHz.

An advantage of utilising an electron beam for the source of chargedparticles is that the whole instrument can be built on a platformcontaining an electron microscope. Accordingly, the present inventionprovides an apparatus further comprising an electron microscope.Accordingly, the present invention provides an apparatus wherein thesource of charged particles is an electron beam wherein the electronbeam is an electron source in the electron microscope.

Charged Particle Column

The charged particle column directs the charged particles to the sample.The charged particle column comprises: a mass filter in order to filterout impurities in the charged particle beam; lenses and apertures asappropriate in order to control the intensity and shape of the primaryion beam; and deflection plates in order to shape the primary ion beamand optionally raster the charged particle beam across the surface ofthe sample (Villacob, 2016). Ion lenses and other components forconstructing the charged particle column are commercially available,e.g. from Agilent. Accordingly, the charged particle column of thepresent invention can provide a charged particle beam scanning systemadapted to scan the beam of charged particles across a plurality oflocations on the sample stage

Typically, the charged particle beam used for secondary ion generationherein has a spot size (i.e. size of the charged particle beam when ithits the sample) of 100 μm or less, such as 20 μm or less, 5 μm or less,1 μm or less, or 500 nm or less, or 300 nm or less, 200 nm or less, 100nm or less, 50 nm or less, or 30 nm or less. The distance referred to asspot size corresponds to the longest internal dimension of the ion beam,e.g. for a circular beam it is a beam of diameter 2 μm, for a squarebeam corresponds to the length of the diagonal between opposed corners,for a quadrilateral it is the length of the longest diagonal etc. Beamshaping and beam masking can be employed to provide the spot shape andsize.

When used for analysis of biological samples, in order to analyseindividual cells, the spot size of ion beam used will depend on the sizeand spacing of the cells. For example, where the cells are tightlypacked against one another (such as in a tissue section) the chargedparticle beam can have a spot size which is no larger than these cellsif single cell analysis is to be conducted. This size will depend on theparticular cells in a sample, but in general the ion beam spot will havea diameter of less than 4 μm e.g. within the range 0.1-4 μm, 0.25-3 μm,or 0.4-2 μm. Thus, a charged particle beam spot can have a diameter ofabout 3 μm or less, about 2 μm or less, about 1 μm or less, about 0.5 μmor less than 0.5 μm, such as about 400 nm or less, about 300 nm or less.In particular embodiments, the spot size is about 200 nm or less, about100 nm or less than 100 nm. In order to analyse cells at a subcellularresolution the system uses a primary ion beam spot size which is nolarger than these cells, and more specifically uses a primary ion beamspot size which can ablate material with a subcellular resolution.Sometimes, single cell analysis can be performed using a spot sizelarger than the size of the cell, for example where cells are spread outon the slide, with space between the cells. The particular spot sizeused can therefore be selected appropriately dependent upon the size ofthe cells being analysed. In biological samples, the cells will rarelyall be of the same size, and so if subcellular resolution imaging isdesired, the ion beam spot size should be smaller than the smallestcell, if constant spot size is maintained throughout the sputteringprocedure.

Lasers

In the present invention, lasers can be used in order to ablatematerial, or to assist in sputtering of material by illuminating alocation after a pulse of charged particles has been passed towards alocation on a sample (see for example, FIGS. 7 and 8 and accompanyingdescription). In addition, lasers can be used to ionise sputteredmaterial previously sputtered by a beam of charged particles (see forexample, FIGS. 6 and 9 and accompanying description). Furthermore,lasers can be used for a combination of the above, see for example FIG.10 and accompanying description.

As the skilled person will appreciate, the requirements of a laser forablating/assisting the sputtering of material differ from therequirements of a laser for ionising material. Generally, lasers forassisting in the sputtering of material or for ablation provide energiesof the order of μJ for pulse lengths of the order of 300 to 400 fs orgreater. In contrast, lasers for ionising sputtered material are morepowerful, providing energies of the order of 1 mJ or greater for pulselengths shorter than 100 fs. The apparatus of the present invention mayuse different lasers for the first and second laser source or may useone laser for the first and second laser source and a beam splitter tosplit the laser and provide the first and second laser sources.

A variety of different lasers can be used for SNMS, including commerciallasers as discussed above in relation to the laser of the laser ablationsampling system, adapted as appropriate to enable sputtering ofmaterial. Generally, the laser is operated at the maximum achievableintensity in order to maximise ionisation. Various types of laser whichcan be used with the present invention are described below. Femtosecondlasers as discussed above are also advantageous in particular SNMSapplications. For example, in the embodiments of the invention set outabove which include first and second laser sources, a single femtosecondlaser can be used to provide both laser sources. The laser could have arepetition rate of 1 MHz and a pulse width of 200 fs with the energypulse of 1 microJ. The skilled person would understand that abeamsplitter can be used to split the laser beam of the laser to providethe first and second laser sources of the claimed invention.

Generally, lasers useful for ionisation include those which supplyenergy on the scale of a few microJoules of energy per pulse, but whichare capable of producing those pulses with high frequency. The laser isfor generating elemental ions from the sample material. The elementalions generated can then be analysed by the mass spectrometer in theapparatus. The laser may be a picosecond laser or a femtosecond laser,or a nanosecond laser. In some embodiments the laser is a femtosecondlaser.

The femtosecond laser may be a solid state laser. Passively mode-lockedsolid-state bulk lasers can emit high-quality ultrashort pulses withtypical durations between 30 fs and 30 μs. Examples of such lasersinclude diode-pumped lasers, such as those based on neodymium-doped orytterbium-doped crystals. Titanium—sapphire lasers can be used for pulsedurations below 10 fs, in extreme cases down to approximately 5 fs (e.g.Octavius Ti:Sapphire Lasers, available from Thorlabs). The pulserepetition rate is in most cases between 1 kHz and 500 MHz.

The femtosecond laser may be a fiber laser. Various types of ultrafastfiber lasers, which may also be passively mode-locked, typically offerpulse durations between 50 and 500 fs, repetition rates between 0.10 and100 MHz, and average power between a few milliwatts and several watts(femtosecond fiber lasers are commercially available from Toptica, IMRAAmerica, Coherent, Inc.).

The femtosecond laser may be a semiconductor laser. Some mode-lockeddiode lasers can generate pulses with femtosecond durations. Directly atthe laser output, the pulses durations are usually at least severalhundred femtoseconds, but with external pulse compression, much shorterpulse durations can be achieved.

In some embodiments, the laser is a nanosecond laser. The nanosecondlaser can be a pumped laser such as the Quantel Q-smart DPSS, the SolarLaser LQ929 high power pulsed Nd:YAG laser, or the Litron High EnergyPulsed Nd:YAG laser. All of these lasers can produce deep ultravioletradiation within the mJ regime so suitable for ionisation and with shortpulse durations.

It is also possible to passively mode-lock vertical external-cavitysurface-emitting lasers (VECSELs); these are interesting particularlybecause they can deliver a combination of short pulse durations, highpulse repetition rates, and sometimes high average output power, whereasthey are not suitable for high pulse energies.

In some embodiments the laser is adapted to produce a pulse ofnanosecond, picosecond or femtosecond scale pulse duration. For example,the laser may have a duration of 500 fs or less, such as 400 fs or less,300 fs or less, 200 fs or less, 100 fs or less, 50 fs or less, 45 fs orless, 25 fs or less, 20 fs or less or 10 fs or less. A femtosecond laseris adapted to produce pulses with a duration of less than 1 μs.

In some embodiments, the laser is adapted to have a pulse repetitionrate of at least 100,000 Hz, such as at least 1 MHz, at least 2 MHz, atleast 3 MHz, at least 4 MHz, at least 5 MHz, at least 10 MHz, at least20 MHz, at least 50 MHz, at least 100 MHz, at least 200 MHz, at least500 MHz or 1 GHz or more.

In some embodiments, the laser is adapted to have beam width (1/e²) of50 μm or less, 20 μm or less, 10 μpm or less, or 5 μm or less. The focalpoint of the laser is where the beam's energy is most concentrated andaccordingly where the greatest ionisation is achieved.

In some embodiments, the laser is adapted to have a pulse energy ofbetween 1 nanoJoule up to 50 milliJoules. Lasers for assisting in thesputtering of material or for ablation of material can be adapted tohave a pulse energy of between 1 nanoJoule and 100 microJoules, such asbetween 10 nanoJoules and 100 microJoules, between 100 nanoJoules and 10microJoules, between 500 nanoJoules and 5 microJoules, such as around 1microJoule, around 2 microJoules, around 3 microJoules or around 4microJoules. Laser for post ionisation can be adapted to have a pulseenergy of between 1 milliJoule and 50 milliJoules, such as between 5milliJoules and 40 milliJoules, 10 milliJoules and 30 milliJoules, 20milliJoules and 35 milliJoules, or around 25 milliJoules or 35milliJoules.

In some embodiments, the laser is adapted to have a pulse energy ofaround 1 microJoule, to have a pulse repetition rate of at least 10 MHz,and to produce pulses with a duration of less than 100 fs, such as 50 fsor less, 45 fs or less, 25 fs or less, 20 fs or less or 10 fs or less.

As the skilled person will appreciate, the present invention can be usedwith one or a combination of the suitable lasers as described herein.For completeness however, a number of specific combinations of laserswhich can be used with present invention are as follows:

Laser for assisting in the sputtering of material or for ablation ofmaterial Laser for post ionisation 2nd harmonic 532 nm Nd:Yag laser 355nm laser (Spectra Physics GCR 150) (CNI Laser, PS-R-355) 2nd harmonic of283.3 nm dye 283.8 nm laser laser pumped by another Nd:Yag (SpectraPhysics GCR 230) laser (Spectra Physics GCR 230), average power oflasers 0.3 mW and 12 mW respectively Excimer laser KrF 248 nm Excimerlaser ArF 193 nm (AZO Optics) (ThorLabs NM07-H01)

Post-ionisation requires a high energy density from the laser radiationin a small volume, e.g. 5 μm³ or less. Because the post ionizationvolume is so small it sets the limit on the amount of material that canbe ionized in one go. If a large amount of positive and negative chargesis created in a small volume the motion of the ions formed will bedominated by the local fields resulting from the space charge induced bythe ions and electrons. If there are too many charged ions in a smallvolume, external fields, such as the fields from ion optics present inmass spectrometers used to direct the resulting ions to the detector fordetection, will not be effective at separating positive and negativecharges and such ion clouds will eventually neutralise reducingionisation efficiency. For example, an ion cloud on a scale of 10 μm (indiameter) containing 10000 elemental charges creates an electrostaticpotential that is about 3 V. Since a few eV is the energy holding theelectrons to the atoms it is also the likely energy level of freeelectrons after ionisation. As a result, the ion density on the scale of10000 ions in a volume on the 10 micrometer scale is near the limitwhere the space charge behaviour starts to dominate.

Accordingly, such effects can be avoided by ensuring that the amount ofsputtered material is kept reasonably small. For example, ablation ofmaterial on the scale of 10×10×10 nm cube to 30×30×30 nm cube or asimilar volume represents the highest amount of material that can betransferred into the post ionization area of a few micrometers is sizewithout creating a strong space change and ion neutralization. Since thesystem can only process 30×30×30 nm cube per single event this createsan opportunity to conduct the imaging with the spatial resolution of 30nm or even 10 nm, to which ion beams can be focused (as discussedherein).

The post ionization beam is co-aligned with the sputtering beam towithin a micrometer precision, as is commonly obtained in opticalsetups.

In some embodiments, the laser beam is directed at an angle to thesample and into the previously ablated area of the specimen (see FIG.6). This configuration minimizes the interaction between the laser lightand the unablated specimen. The laser beam can be focused to a tightfocus that overlaps with the volume of ablation plume. The laser beamfocusing can be done at high numerical aperture (NA) to facilitate sharpfocusing in the overlapping region and enable rapid laser energyspreading outside of the overlapping region to minimize the possibilityof damage to the sample in the regions surrounding that being sampled.

Due to constraints in FIG. 6 of the ion optics that focuses the primarybeam and transfers the secondary beam for mass analysis, the space forthe laser beam optics may be limited in some embodiments. As a result,in some embodiments, numerical aperture of the laser beam may beconstrained in one of the planes. For example, the laser beam may have alow NA in the plane of the drawing to avoid interferences with ionoptics and high NA in the plane perpendicular to the plane of thedrawing. Such an arrangement results in an elliptical focal spot that isextended in the plane of low NA. The elliptical focal spot may improvethe degree of overlap with the sputtered/ablated plume. Accordingly, insome embodiments, the laser of the post-ionisation system has anelliptical focal spot.

In addition to spatial co-ordination, the sputtering due to the primaryion or electron beam needs to be synchronised with the delivery of laserradiation to ionise the sputtered material. The speed at which thesputtered material leaves the target is typically on the scale of thespeed of sound i.e. 1000 m/s. Thus, to ensure alignment of the sputteredcloud and the laser beam with 1 micrometer precision requires timingprecision on the scale of 1 ns. Ion beam bunching and timing to 1 ns iscommonly achieved with accelerating voltages on the order of 5 to 20 kV.The same or similar bunching technology can be applied to the primarybeam. This technology is used, for instance, in current TOF MSapparatus.

Thus in this mode of operation, the primary ion beam or electron beamsputters material from the sample, and very shortly after that, theejected material is post-ionised by a pulse of laser radiation.

To further facilitate consistent sputtering/ejection of material fromthe target, in some instances, the target may be illuminated with apulse of a laser light that is synchronized with the pulse of primaryions (instead of, or in addition to, the post-ionisation discussedabove). The energy of the laser pulse should be set below ablationthreshold for the material that has not been exposed to the primary ionor electron beam. In the area where the primary ion beam or electronbeam interacts with the target, the state of the matter resembles plasmaand since it is confined to a nano-scale it is referred to herein as anano-plasma. Thus, the optical beam can interact with the nano-plasmaand pump additional energy into this volume leading to a consistentdesorption of material. This mode of desorption can be viewed as anignition state (provided by the primary ion beam or electron beaminteracting with the target and creating nano-plasma) followed by anenergy pumping state where the laser light pumps additional energy intothe nano-plasma facilitating its ejection.

This mode of operation can help to overcome the limitation of relativelyslow sputtering by the primary ion or electron beam. Sputtering rate dueto the primary ion beam/electron beam is limited by the total number ofprimary ions/electrons impacting the given area. The focusing ability ofthe primary ion beam/electron beam is limited due to the ion/electroncurrent and space charge effects. This, combined with the need to timethe primary ion beam/electron beam to only sputter material when thepost ionization laser is off places a limit on the sputtering rate. Useof laser radiation to enhance sputtering can overcome the limitation asfewer primary particles are needed to seed the sample material withexcitation energy needed for the laser beam to affect thesputtering/ablation.

In instances where a laser is used both for post-ionization andenhancing sputtering, the same laser could also be suitable for energypumping into the nano-plasma to facilitate material desorption. Forexample, a single femtosecond laser can service both needs in theapparatus. The laser could have a repetition rate of 1 MHz and a pulsewidth of 200 fs with the energy per pulse of 1 microJ (i.e. averagepower of 1 W). Such laser pulse could be split up (e.g. using a beamsplitter) and a portion of it used as is for energy pumping ofnano-plasma. The remaining part could be used for post ionization. Acompression stage can be applied to the post ionization beam to reducethe amount of energy per pulse required for post-ionization.

In some embodiments, the post ionization with the laser beam is carriedout by pumping the energy to the nano-plasma on the surface. Thus, thedesorption pumping and post ionization processes are combined into asingle process activated by the laser pulse delivered to the nano-plasmaon the target.

Moreover, the energy pumping into plasma on the target can beaccomplished by tuning the laser wavelength to the absorption band ofthe plasma while avoiding absorption band of the unionized targetmaterial. As such, the impact of the primary ion beam/electron beamserves to change the state of the matter in the target fromnon-absorbing the pumping light to absorbing the pumping light. Theduration of the pumping light can be on the scale of picoseconds up to afew nanoseconds and down to a few femtoseconds. At the same time, thepicosecond pulse is short enough to avoid significant diffusionbroadening of the plasma volume in the sample.

In some embodiments, the laser radiation for energy pumping is deliveredthrough the sample carrier (see FIG. 8). For example, the primaryelectron beam or the primary ion beam can be focused to a spot on thescale of 10 to 30 nm while the laser light focusing is limited bydiffraction effects and will likely stay on the scale of 100 to 1000 nm.

Equipment known in the art can be used to introduce delay between laserpulses. Accordingly, in some embodiments, the apparatus comprises anoptical delay line to introduce delay between laser pulses. Examples ofoptical delay line suitable for use in the present invention are any ofthe optical delay lines commercially available from ThorLabs.

Variable delay lines can be used to synchronise the arrival of theionisation pulse with respect to the packet of primary ions orelectrons, thus enabling the same laser to act in post-ionisation orpumping modes, depending of the relative timing of the packet of primaryions or electrons and the laser pulse.

Sample Chamber

The sample chamber of the sputtering-based sampling system shares manyfeatures in common with the sample chamber of the laser ablation-basedsampling system discussed above. It comprises a stage to support thesample. The stage may be a translation stage, movable in the x-y orx-y-z axes. The sample chamber will also comprise an outlet, throughwhich material removed from the sample by the laser radiation can bedirected. The outlet is connected to the detector, enabling analysis ofthe sample ions.

In some instances, the sample chamber is held under 133322-13.3 Pa, suchas 1333.22-133.322 Pa. In some instances, the sample chamber is heldunder a vacuum. Accordingly, in some instances, the sample chamberpressure is lower than 50000 Pa, lower than 10000 Pa, lower than 5000Pa, lower than 1000 Pa, lower than 500 Pa, lower than 100 Pa, lower than10 Pa, lower than 1 Pa, around 0.1 Pa or less than 0.1 Pa, such as 0.01Pa or lower. For instance, partial vacuum pressure may be around 50-2000Pa, 100-1000 Pa, or 200-700 Pa, and vacuum pressure lower than 10 Pa, 1Pa, or 0.2 Pa. Suitable gasses include Argon, Helium, Nitrogen andmixtures thereof.

The selection of whether the sample pressure is at atmospheric pressure,partial vacuum pressure, or under vacuum depends on the particularanalysis being performed, as will be understood by one of skill in theart. For instance, at atmospheric pressure, sample handing is easier,and softer ionisation may be applied. Further, the presence of gasmolecules may be desired so as to enable the phenomenon of collisionalcooling to occur, which can be of interest when the label is a largemolecule, the fragmentation of which is not desired, e.g. a molecularfragment comprising a labelling atom or combination thereof.Alternatively, in instances where laser radiation is used to post-ionisematerial, the presence of gas molecules (e.g. at partial vacuumpressure) may be advantageous. For example, collisional cooling mayallow the cooling of the nanoplasma generated at the surface of thesample (e.g., following charged particle bombardment or laserillumination of the sample to generate the energy pumping state) andexpansion of the plume of ablated material and before re-ionization in apost-ionization system. Collisions may also allow for at least partialcharge reduction, reducing space-charge effects and/or improving theability of ion optics to direct ions. Charge state reduction to just asingle charge per ion makes it easier to read out signals for differentmass tags.

Holding the sample chamber under vacuum can prevent collisions betweensample ions generated and other particles within the chamber.

The principal difference between the sample chamber of the SNMS systemand the sample chamber of the laser ablation-based system is that thechamber is held under vacuum in order to prevent collisions betweensample ions and other particles within the chamber, which could resultin loss of charge from the ions—on a similar basis contrary to the laserablation and desorption-based sample chambers. Loss of secondary ionswould result in reduced sensitivity for the apparatus.

Ion Microscope

The sample ions are captured from the sample via an electrostatic lenspositioned near to the sample, known in the art as an immersion lens (oran extraction lens). The immersion lens removes the secondary ionsimmediately from the locality of the sample. This is typically achievedby the sample and the lens having a large difference in voltagepotential. Depending on the polarity of the sample vis-à-vis theimmersion lens, positive or negative secondary ions are captured by theimmersion lens. The polarity of the sample ions as captured by theimmersion lens is independent of the polarity of the ions of the chargedparticle beam.

The sample ions are then transferred to the detector by via one or morefurther electrostatic lenses (known as transfer lenses in the art). Thetransfer lens(es) focus(es) the beam of secondary ions into thedetector. Typically, in systems with multiple transfer lenses, only onetransfer lens is engaged in a given analysis. Each lens may provide adifferent magnification of the sample surface. Commonly, further ionmanipulation components are present between the immersion lens and thedetector, for example one or more apertures, mass filters or sets ofdeflector plates. Together, the immersion lens, transfer lens, and anyfurther components, form the ion microscope. Components for theproduction of an ion microscope are available from commercial supplierse.g. Agilent.

Camera

The system may also comprise a camera. Camera systems are discussedabove in relation to laser ablation sampling systems, and the featuresof the above camera can also be present in the secondary ion generationsystem, except where incompatible (e.g. it can be connected to a lightmicroscope, such as a confocal microscope, but it is not possible tofocus a primary ion beam through the same optics as the light which isdirected to the camera, because one beam is ions and the other photons).

c. Two-Pulse Laser Based Sampling and Ionising System

Akin to certain apparatus, systems and techniques discussed in section1.b. above, electron seeding to generate a ‘sample ignition state’ canbe achieved at a location by a pulse of laser radiation. Subsequent tothis, a second laser pulse with a laser spot diameter greater than thelocation, but with a fluence lower than the ablation threshold of thesample, can be directed at the sample to cause ablation only at thelocation targeted with the first laser pulse, as the energy needed toablate the pre-seeded location is lower than the surrounding area. Thus,a tightly focused, low energy, first pulse can be used can be used toseed electrons, and a higher energy, less focused, pulse can be used tocause ablation at the location at which electrons were seeded. Thus, theinvention provides yet a further means for high resolution imaging basedupon this technique.

It is well known optical behaviour that the minimum spot size generatedby an optical system is directly proportional to the wavelength of thelight and inversely proportional to the numerical aperture of theobjective. Since the wavelength of visible light is around 500 nm andnumerical aperture of typical objectives rarely exceeds 1.0, the size ofthe focusing spot with such light ends up just below one micrometer.Accordingly, by operating with a shorter wavelength laser, the light canbe focused to a smaller spot on the sample. For example, a VUV lightwith a wavelength around 200 nm can be generated by excimer lasers.Moreover, modern lithography tools employ EUV light with a wavelength of13.5 nm. Unfortunately, generation of VUV and EUV light pulse with theenergy sufficient for ablation requires a large, complex and expensiveapparatus.

The invention solves the problem of ablation spot reduction bypre-seeding the spot to be ablated with a small amount of UV, EUV or XUVlight. This pre-seeding generates free electrons in the otherwisedielectric material. This change in material properties lowers thethreshold for ablation by a subsequent pulse. The amount of energyneeded for the pre-seeding is many orders of magnitude lower than theamount of energy needed for direct ablation. This makes the equipmentfor generating such pulse relatively economical. The pre-seeding pulsecan be focussed to 100 or 30 nm diameter because of its shortwavelength. Shortly after the pre-seeding is done a pulse with a morecommon wavelength such as IR or Visible is sent to the same sample. Itdeposits its energy much more efficiently in the pre-seeded area. As aresult, the pre-seeded area can be ablated while the rest of the areacovered by the IR light won't be ablated. Thus, the size of ablationspot is controlled by the diffraction limit imposed by the UV, VUV, EUVwavelength. This limit is substantially lower than the ablationresolution of a Visible or IR light applied on its own.

In addition, this invention can lead to ionization of ablated material.Ion composition of ablated material can be analyzed and provide imagingmass spectrometry or imaging mass cytometry workflow. The material beingablated will be ionized in a plasma leading to a significant fraction ofelemental ions being generated. Efficiency of ionization and ionsampling from the plasma can become high enough to facilitate singlecopy detection of antibodies when utilizing MaxPar reagents or similarreagents. Here, the ions can be extracted directly for analysis via theion microscope as discussed below.

Several analytical problems exist when operation at such a high spatialresolution (e.g. 30 nm). One problem is the total acquisition rate vsthe field of view (or region of interest) dimensions. An existingHyperion operates at 200 pixels/s with 1 micrometer pixel size. Thisresults in ˜5000 seconds needed to record an image of a biologicallyrelevant ROI with 1×1 mm scale. If the pixel size is reduced 30 times(to ˜30 nm) then the amount of time would grow to 500000 s to record animage from a similar area. It is obviously, an impractically long time.This invention solves this problem by operating ionization sampling inat least a partial vacuum (see discussion of sample chamber below forfurther details) where the pixel rate can be as high as 1 Mpixels/swhich means the 1×1 mm area will be acquired in just 100 seconds.Multiple serial sections can be analyzed to provide 3D images on abiological scale of around 0.03 mm. It will take 1000 sections (30 nmeach) and ˜1 day of experiment. This is a very high speed consideringthe multiparametric and high spatial resolution nature of the dataobtained. If acquisition rate is increased to 10 Mpixels/s thecollection of such images will be permitted within 2.5 hours. Sampleanalysis will also go ˜30× faster at the spatial resolution of 100 nm.

As noted above, the use of MaxPar reagents (mass tagged antibodyreagents, as discussed below) can compensate for sensitivity loss due tothe smaller ablations by having ˜100 atoms per a copy of one antibody.Ion sampling and transmission above 5% enables detection of a singlecopy of antibody. When an object is imaged with a pixel size of 30 nmonly a very few antibodies can be found in such pixel. Therefore, itbecomes vital to enable single copy detection for imaging at such asmall scale.

The plasma generated by the two-pulse technique of the invention cansolve the problem of efficient ionization and sampling. The small scaleof ablation leads to the small scale of plasma which in turn reduces theprobability of plasma neutralization during ion sampling. Moreover, thedensity of the solid material being ablated leads to initial plasmapressure on the scale 10000 atm which in turn leads to a local thermalequilibrium model of the plasma. The local thermal equilibrium permitsgeneration of an optimum plasma temperature to facilitate near 100%efficient ionization of labelling atoms from mass tags to form elementalions, controllable via the parameters of the second pulse.

A two-pulse laser ablation system based apparatus typically comprisesthree components. The first component is a first laser source forseeding electrons in a sample (which would be housed on the sample stagein the apparatus and systems disclosed herein). The second component isa second laser source for ablating the sample where electrons have beenseeded by the first laser source. (Together, these two components form atwo-pulse laser sampling and ionisation system.) The third component isa detector component that detects the ionised material, for instance amass detector. The laser sources are typically pulsed. In someinstances, the first (pre-seeding) pulse can be derived from the samelaser that delivers the second (ablating) pulse, for instance by meansof harmonics generation. The mentions in this section of directing alaser beam towards/at a location on the sample stage refers to thearrangement of the components in the apparatus and system, but as willbe appreciated by one in the art, when the apparatus/system is beingused to analyse a sample, the laser radiation from the first and secondlaser sources will impinge upon the sample under analysis.

Accordingly, the invention provides an apparatus for analysing a sample,such as a biological sample, comprising:

-   -   a sample stage;    -   a first laser source, configured to seed electrons in a sample,        and first focusing optics configured to direct a laser beam        emitted by the first laser source towards the sample stage; and    -   a second laser source, configured to ablate sample material        pre-seeded with electrons by the first laser source, and second        focusing optics configured to direct a laser beam emitted by the        second laser source towards the sample stage.

The apparatus typically comprises a mass detector, such as a TOFdetector.

Accordingly, the invention provides a two-pulse laser sampling andionisation system for analysing a sample, such as a biological sample,comprising:

-   -   a sample stage;    -   a first laser source, configured to seed electrons in a sample,        and first focusing optics configured to direct a laser beam        emitted by the first laser source towards the sample stage; and    -   a second laser source, configured to ablate sample material        pre-seeded with electrons by the first laser source, and second        focusing optics configured to direct a laser beam emitted by the        second laser source towards the sample stage.

In some embodiments, the first focusing optics configured to direct alaser beam emitted by the first laser source towards the sample stage isconfigured to direct a laser beam emitted by the first laser source to alocation on the sample stage, and the second focusing optics configuredto direct a laser beam emitted by the second laser source towards thesample stage is configured to direct a laser beam emitted by secondfirst laser source to the location on the sample stage.

For instance, the invention provides an apparatus for analysing asample, such as a biological sample, comprising:

-   -   a sample stage;    -   a first laser source, configured to seed electrons in a sample,        and first focusing optics configured to direct a laser beam        emitted by the first laser source towards a location on the        sample stage; and    -   a second laser source, configured to ablate sample material        pre-seeded with electrons by the first laser source, and second        focusing optics configured to direct a laser beam emitted by the        second laser source towards the location on the sample stage.

The apparatus typically comprises a mass detector, such as a TOFdetector.

For instance, the invention provides a two-pulse laser sampling andionisation system for analysing a sample, such as a biological sample,comprising:

-   -   a sample stage;    -   a first laser source, configured to seed electrons in a sample,        and first focusing optics configured to direct a laser beam        emitted by the first laser source towards a location on the        sample stage; and    -   a second laser source, configured to ablate sample material        pre-seeded with electrons by the first laser source, and second        focusing optics configured to direct a laser beam emitted by the        second laser source towards the location on the sample stage.

FIG. 13 is a schematic diagram of the arrangement of an exemplaryembodiment of the invention that demonstrates reduction of the ablationspot size by two pulse ablation. A support target 20 holds a thinsection of the specimen 30. In some embodiments (e.g. nano-machiningapplications) the support target and the specimen can be one body. Apulse of UV, or VUV or EUV or even XUV light 40 is focused 50 on thespecimen 30 by an objective 60. Special objectives for UV, VUV, EUVoptics are known in the art and are often based on reflective opticalarrangements. The pulse of e.g. EUV light creates a seed of freeelectrons in the focal spot. Since the sample is generallynon-conductive, pre-seeding with free electrons alters the properties onmaterial in the focal spot of the laser. A second pulse of light 70 issent to the same area, encompassing the location in which electrons werepre-seeded, to supply the energy to develop plasma in the preseededlocation. The second pulse of light 70 is sent by another objective 80.Due to a significantly longer wavelength of the second pulse the focalspot 90 of the second pulse is much larger than the focal spot of thefirst pulse. The process by which electrons multiply in the specimenduring the second pulse can be called avalanche ionization. In practice,many phenomena can contribute to the preferential excitation of thepre-seeded area. As long as the energy required for the preferentialexcitation is substantially lower than the threshold for direct ablationthe scheme of defining ablation zone by the pre-seeding with the e.g.EUV pulse is effective.

Accordingly, the invention provides an apparatus and systems foranalysing a sample, such as a biological sample, wherein the secondfocusing optics is configured to synchronise a second pulse, of laserradiation from the second laser source, to arrive at the location on thesample stage directly after a pulse of laser radiation from the firstlaser source

Accordingly, the invention provides an apparatus for analysing a sample,such as a biological sample, comprising:

-   -   a sample stage;    -   a first laser source, configured to seed electrons in a sample,        and first focusing optics configured to direct a laser beam        emitted by the first laser source towards the sample stage; and    -   a second laser source, configured to ablate sample material        pre-seeded with electrons by the first laser source, and second        focusing optics configured to direct a laser beam emitted by the        second laser source towards the sample stage;    -   wherein the second focusing optics is configured to synchronise        a second pulse, of laser radiation from the second laser source,        to arrive at the location on the sample stage directly after a        pulse of laser radiation from the first laser source,

The apparatus typically comprises a mass detector, such as a TOFdetector.

Accordingly, the invention provides a two-pulse laser sampling andionisation system for analysing a sample, such as a biological sample,comprising:

-   -   a sample stage;    -   a first laser source, configured to seed electrons in a sample,        and first focusing optics configured to direct a laser beam        emitted by the first laser source towards the sample stage; and    -   a second laser source, configured to ablate sample material        pre-seeded with electrons by the first laser source, and second        focusing optics configured to direct a laser beam emitted by the        second laser source towards the sample stage;    -   wherein the second focusing optics is configured to synchronise        a second pulse, of laser radiation from the second laser source,        to arrive at the location on the sample stage directly after a        pulse of laser radiation from the first laser source,

In some embodiments, the first focusing optics configured to direct alaser beam emitted by the first laser source towards the sample stage isconfigured to direct a laser beam emitted by the first laser source to alocation on the sample stage, and the second focusing optics configuredto direct a laser beam emitted by the second laser source towards thesample stage is configured to direct a laser beam emitted by secondfirst laser source to the location on the sample stage.

Accordingly, the invention provides an apparatus and systems foranalysing a sample, such as a biological sample, wherein the firstfocusing optics and second focusing optics are configured to directlaser radiation to opposite sides of the sample stage.

Accordingly, the invention provides an apparatus for analysing a sample,such as a biological sample, comprising:

-   -   a sample stage comprising a first face and a second face, the        first and second faces being opposed, and wherein the first face        is adapted to receive a sample;    -   a first laser source, configured to seed electrons in a sample,        and first focusing optics configured to direct a laser beam        emitted by the first laser source towards the first face of the        sample stage; and    -   a second laser source, configured to ablate sample material        pre-seeded with electrons by the first laser source, and second        focusing optics configured to direct a laser beam emitted by the        second laser source towards the second face of the sample stage;    -   wherein the second focusing optics is configured to synchronise        a second pulse, of laser radiation from the second laser source,        to arrive at the location on the sample stage directly after a        pulse of laser radiation from the first laser source.

The apparatus typically comprises a mass detector, such as a TOFdetector.

Accordingly, the invention provides a two-pulse laser sampling andionisation system for analysing a sample, such as a biological sample,comprising:

-   -   a sample stage comprising a first face and a second face, the        first and second faces being opposed, and wherein the first face        is adapted to receive a sample;    -   a first laser source, configured to seed electrons in a sample,        and first focusing optics configured to direct a laser beam        emitted by the first laser source towards the first face of the        sample stage; and    -   a second laser source, configured to ablate sample material        pre-seeded with electrons by the first laser source, and second        focusing optics configured to direct a laser beam emitted by the        second laser source towards the second face of the sample stage;    -   wherein the second focusing optics is configured to synchronise        a second pulse, of laser radiation from the second laser source,        to arrive at the location on the sample stage directly after a        pulse of laser radiation from the first laser source.

The invention also provides an apparatus for analysing a sample, such asa biological sample, comprising:

-   -   a sample stage comprising a first face and a second face, the        first and second faces being opposed, and wherein the first face        is adapted to receive a sample;    -   a first laser source, configured to seed electrons in a sample,        and first focusing optics configured to direct a laser beam        emitted by the first laser source towards the second face of the        sample stage; and    -   a second laser source, configured to ablate sample material        pre-seeded with electrons by the first laser source, and second        focusing optics configured to direct a laser beam emitted by the        second laser source towards the first face of the sample stage;    -   wherein the second focusing optics is configured to synchronise        a second pulse, of laser radiation from the second laser source,        to arrive at the location on the sample stage directly after a        pulse of laser radiation from the first laser source.

The apparatus typically comprises a mass detector, such as a TOFdetector.

Accordingly, the invention provides a two-pulse laser sampling andionisation system for analysing a sample, such as a biological sample,comprising:

-   -   a sample stage comprising a first face and a second face, the        first and second faces being opposed, and wherein the first face        is adapted to receive a sample;    -   a first laser source, configured to seed electrons in a sample,        and first focusing optics configured to direct a laser beam        emitted by the first laser source towards the second face of the        sample stage; and    -   a second laser source, configured to ablate sample material        pre-seeded with electrons by the first laser source, and second        focusing optics configured to direct a laser beam emitted by the        second laser source towards the first face of the sample stage;    -   wherein the second focusing optics is configured to synchronise        a second pulse, of laser radiation from the second laser source,        to arrive at the location on the sample stage directly after a        pulse of laser radiation from the first laser source.

FIG. 14 shows another preferred embodiment for invention. Here, thefirst pulse 40 and the second pulse 70 are focused onto the specimenfrom the same side. A single objective 60 is used to combine and focustwo light pulses. Accordingly, the invention provides an apparatus andsystems for analysing a sample, such as a biological sample, wherein thefirst focusing optics and second focusing optics are configured todirect laser radiation to opposite sides of the sample stage.

Accordingly, the invention provides an apparatus for analysing a sample,such as a biological sample, comprising:

-   -   a sample stage comprising a first face and a second face, the        first and second faces being opposed, and wherein the first face        is adapted to receive a sample;    -   a first laser source, configured to seed electrons in a sample,        and first focusing optics configured to direct a laser beam        emitted by the first laser source towards the first face of the        sample stage; and    -   a second laser source, configured to ablate sample material        pre-seeded with electrons by the first laser source, and second        focusing optics configured to direct a laser beam emitted by the        second laser source towards the first face of the sample stage;    -   wherein the second focusing optics is configured to synchronise        a second pulse, of laser radiation from the second laser source,        to arrive at the location on the sample stage directly after a        pulse of laser radiation from the first laser source;    -   and wherein the first focusing optics and second focusing optics        use the same objective lens to focus radiation from the first        and second laser sources.

The apparatus typically comprises a mass detector, such as a TOFdetector.

Accordingly, the invention provides a two-pulse laser sampling andionisation system for analysing a sample, such as a biological sample,comprising:

-   -   a sample stage comprising a first face and a second face, the        first and second faces being opposed, and wherein the first face        is adapted to receive a sample;    -   a first laser source, configured to seed electrons in a sample,        and first focusing optics configured to direct a laser beam        emitted by the first laser source towards the first face of the        sample stage; and    -   a second laser source, configured to ablate sample material        pre-seeded with electrons by the first laser source, and second        focusing optics configured to direct a laser beam emitted by the        second laser source towards the first face of the sample stage;    -   wherein the second focusing optics is configured to synchronise        a second pulse, of laser radiation from the second laser source,        to arrive at the location on the sample stage directly after a        pulse of laser radiation from the first laser source; and    -   wherein the first focusing optics and second focusing optics use        the same objective lens to focus radiation from the first and        second laser sources.

FIG. 15 shows yet another embodiment where the sample (30) is resting ona material that is at least semi-transparent to the first pulse and evenmore transparent to the second pulse. This material may be in the formof a metal mesh used in electron microscopy. This geometry allows forthe ablated plasma to expand into a cloud 100 of ions, electrons andneutral material. The ions here can be sampled into a mass spectrometerfor imaging mass spectrometry an imaging mass cytometry applications.

Accordingly, in some embodiments of the two-pulse laser based apparatusand sampling and ionisation system disclosed herein, the sample stage isat least in part composed of a material at least semi-transparent to thelaser radiation from the first laser source and even more transparent toradiation from the second laser source. Alternatively, as explainedelsewhere herein, and reiterated here for completeness, when the laserradiation is directed through the sample stage to reach a sample on thefirst face, the sample stage should include a void through which laserradiation can pass to reach the sample (through the sample carrier). Inthe situation where the sample stage comprises a void, the sample isplaced on a sample carrier that is at least in part composed of amaterial at least semi-transparent to radiation from the first lasersource and even more transparent to radiation from the second lasersource.

FIG. 16 shows another embodiment that facilitates efficient sampling ofablated ions. Here the first laser pulse 40 is sent from the first side.The first laser pulse is formed by an objective 60 that has an openingin the middle to allow for the passage of ions 100 generated after theablation.

Accordingly, in some embodiments of the two-pulse laser based apparatusand sampling and ionisation system disclosed herein, the objective ofthe first focusing optics has an opening in the middle to allow for thepassage of ionized material from a sample through the opening.

Components of Two-Laser Sampling and Ionisation System

First Laser Source—Lasers for Pre-Seeding Electrons

As set out above, the requirement to achieve high resolution imaging isa wavelength of laser radiation that can be focused to a focal spot of100 nm diameter or smaller. Accordingly, the wavelength of laserradiation emitted by the first laser source must be UV light. In someembodiments, the laser radiation emitted by the first laser source isUV, VUV, EUV or XUV. In some embodiments, the laser radiation emitted bythe first laser source is 200 nm or shorter, 175 nm or shorter, 150 nmor shorter, 125 nm or shorter, 100 nm or shorter, 75 nm or shorter, 50nm or shorter, 25 nm or shorter, 20 nm or shorter or 15 nm. Forinstance, in some embodiments, the laser radiation emitted by the firstlaser source is between 10-200 nm, between 10-175 nm, between 10-150 nm,between 10-125 nm, between 10-100 nm, between 10-75 nm, between 10-50nm, between 10-25 nm, between 10-20 nm, or between 10-15 nm.

In line with the discussions above, the pulse energy of the first lasersource is chosen such that it seeds electrons in a material but does notablate the material. In some embodiments, the pulse energy of the firstlaser source is in the picoJ to femtoJ range. For instance between 1picoJ-100 femtoJ, between 10 picoJ-100 femtoJ, or between 100 picoJ-1femtoJ.

The duration of the pulse must be short enough to minimise diffusion ofthe seeded electrons prior to the second laser pulse that causesablation of the sample. The total time between the start of the firstpulse (from the first laser source) and end of the second (from thesecond laser source) should be kept below 10 ps. Accordingly, in someembodiments, the duration of a pulse from the first laser source is 5 μsor shorter, such as 2 μs or shorter, 1 μs or shorter, 500 fs or shorter,400 fs or shorter, 300 fs or shorter, 200 fs or shorter or 100 fs orshorter, 50 fs or shorter, 40 fs or shorter, 30 fs or shorter, 20 fs orshorter or 10 fs or shorter.

First Focusing Optics

The first focusing optics direct the radiation from the first lasersource to the sample, and focus it onto the sample. To achieve highresolution imaging, the first focusing optics must focus the laserradiation to a spot size at the sample of around 100 nm or shorter, suchas 75 nm or shorter, 50 nm or shorter or around 30 nm.

In some embodiments, the objective lens of the first focusing optics isa reflective objective.

Second Laser Source—Lasers for Ablation of Pre-Seeded Sample Locations

The requirements for the second laser source in the system differbecause of its different function. The pulse of laser radiation from thesecond laser source supplies the energy to develop the plasma at thesample and to control its temperature.

Accordingly, the wavelength of laser radiation emitted by the secondlaser source can be IR or visible light. If visible light is emitted bythe second laser source, it can be focused tighter and will thereforerequire less energy per pulse for ablation. In some embodiments, thelaser radiation emitted by the second laser source is 400 nm or longer,500 nm or longer, 600 nm or longer, 700 nm or longer, 800 nm or longer,or 1 μm or longer. For instance, in some embodiments, the laserradiation emitted by the second laser source is between 400 nm-100 μm,such as between 200 nm-100 μm, between 200 nm-10 μm, between 200 nm-1μm, between 400 nm-10 μm, between 400 nm-1 μm, between 400 nm-900 nm,between 400 nm-800 nm, between 400 nm-700 nm, between 400 nm-600 nm orbetween 500 nm-600 nm.

In line with the discussions above, the pulse energy of the second lasersource is chosen such that it ablates only the sample material in thelaser spot that has been pre-seeded with electrons—i.e. the pulsefluence is below the ablation threshold of the material. In someembodiments, the pulse energy of the second laser source is in thenanoJoule range. For instance between 1 nanoJ-1 pJ, between 10 nanoJ-500nanoJ, between 50 nanoJ-250 nanoJ, or around 100 nanoJ.

The duration of the pulse must be short enough to minimise diffusion ofthe seeded electrons prior to the second laser pulse that causesablation of the sample. The total time between the start of the firstpulse (from the first laser source) and end of the second (from thesecond laser source) should be kept below 10 μs. Accordingly, in someembodiments, the duration of a pulse from the second laser source is 5μs or shorter, such as 2 μs or shorter, 1 μs or shorter, 500 fs orshorter, 400 fs or shorter, 300 fs or shorter, 200 fs or shorter or 100fs or shorter, 50 fs or shorter, 40 fs or shorter, 30 fs or shorter, 20fs or shorter or 10 fs or shorter.

Second Focusing Optics

The second focusing optics direct the radiation from the second lasersource to the sample, and focus it onto the sample. High resolutionimaging is achieved by the tightness of the focusing of the first shortwavelength pulse, accordingly the spot size may be bigger than the firstpulse. Nonetheless, a relatively small spot size minimises theirradiation of sample around the seeded location. Accordingly, in someembodiments, the second focusing optics focus the laser radiation to aspot size at the sample of around 2 μm or shorter, such as 1 μm orshorter, 750 nm or shorter, 500 nm, 250 nm or shorter, 200 nm orshorter, 150 nm or shorter or around 100 nm.

In some embodiments, the objective lens of the second focussing opticsis a refractive or reflective objective, such as a lens with an NA above0.7, above 0.8 or above 0.9. In some embodiments, the objective lens ofthe second focussing optics is also the objective lens of the firstfocussing optics as discussed above.

Synchronisation of First and Second Laser Sources

The apparatus of the present invention may use different lasers for thefirst and second laser source. Here, techniques routine in the art, suchas programmed module comprising instructions can be used to co-ordinatedelivering of a first pulse, from the first laser source, and a secondpulse, from the second laser source, to the sample, such thatpre-seeding and ablation of the pre-seeded area occur in line with thediscussions above. In some embodiments, the first and second pulses aresynchronised such that the time elapsed from the start of the pulse fromthe first laser source to the end of the pulse from the second lasersource has a duration less than 50 ps, such as shorter than 25 ps. Insome embodiments, the time is shorter than 10 ps, such as shorter than 5ps, shorter than 2 ps, or shorter than 1 ps,

In some embodiments, the apparatus and systems use one laser for thefirst and second laser source and a beam splitter to split the laser andprovide the first and second laser sources.

For instance, a single laser engine that generates an IR pulse can beused. The pulse can be converted into visible light by a second or thirdharmonic generator. To generate short wavelength radiation for the firstpulse, the laser can be coupled into a high-harmonics generation stage.Optical delay lines can be employed control time separation between thefirst and the second pulse as well as beyond the second and the thirdpulse.

Accordingly, in some embodiments, the apparatus or sampling systemcomprising a first laser source and a second laser source comprises asingle laser, beam splitter, and two harmonics generators, wherein oneof the harmonics generators is adapted to produce UV, VUV, EUV or XUVlaser radiation (as discussed above for the first laser source) from thesingle laser as a first laser source, and the other of the harmonicsgenerators IR or visible wavelength laser radiation (as discussed abovefor the second laser source) from the single laser as a second lasersource.

Accordingly, in some embodiments, the apparatus or sampling systemcomprising a first laser source and a second laser source comprises asingle laser, beam splitter, two harmonics generators, and an opticaldelay line, wherein one of the harmonics generators is adapted toproduce UV, VUV, EUV or XUV laser radiation (as discussed above for thefirst laser source) from the single laser as a first laser source, andthe other of the harmonics generators is adapted to produce IR orvisible wavelength laser radiation (as discussed above for the secondlaser source) from the single laser as a second laser source; andwherein the optical delay line is adapted so as to deliver a pulse fromthe second laser source derived from a pulse of the single laser afterthe pulse from the first laser source derived from the same pulse of thesingle laser, such that the end of the second pulse is shorter than 50ps following the start of the first pulse, such as shorter than 25 ps,shorter than 10 ps, such as shorter than 5 ps, shorter than 2 ps, orshorter than 1 ps.

Optionally, the output of the harmonics generator adapted to produce UV,VUV, EUV or XUV laser radiation can be filtered out to contain the rangeof the wavelengths that are of interest for pre-seeding the area to beablated.

A magnetic sector instrument is probably more suited for high rate ofrecording of 1 Mpixel and above. For the rate of recording of 100kpixels/s a Time-Of-Flight mass analyzer is suitable and might besimpler to design and build.

Because the ion source of this type produces a very tight ion spot theproperties of the ion beam in this technique make the ion beam suitablefor many types of mass analyzers (known at present and to be invented).

General Laser Considerations

The femtosecond laser may be a solid state laser. Passively mode-lockedsolid-state bulk lasers can emit high-quality ultrashort pulses withtypical durations between 30 fs and 30 ps. Examples of such lasersinclude diode-pumped lasers, such as those based on neodymium-doped orytterbium-doped crystals. Titanium—sapphire lasers can be used for pulsedurations below 10 fs, in extreme cases down to approximately 5 fs (e.g.Octavius Ti:Sapphire Lasers, available from Thorlabs). The pulserepetition rate is in most cases between 1 kHz and 500 MHz.

The femtosecond laser may be a fiber laser. Various types of ultrafastfiber lasers, which may also be passively mode-locked, typically offerpulse durations between 50 and 500 fs, repetition rates between 0.10 and100 MHz, and average power between a few milliwatts and several watts(femtosecond fiber lasers are commercially available from Toptica, IMRAAmerica, Coherent, Inc.). Femtosecond fiber lasers are particularlysuitable for this application. The lasers are reasonably priced when theenergy output is <1 uJ. The repetition rate of laser pulses is >1 MHzpotentially leading to the acquisition rate of 1 Mpixel/s. The pulseduration of such lasers ˜200 fs is well within the upper limit of 10 μsdictated by diffusion broadening of the plasma area.

The femtosecond laser may be a semiconductor laser. Some mode-lockeddiode lasers can generate pulses with femtosecond durations. Directly atthe laser output, the pulses durations are usually at least severalhundred femtoseconds, but with external pulse compression, much shorterpulse durations can be achieved.

In some embodiments, the laser is a nanosecond laser. The nanosecondlaser can be a pumped laser such as the Quantel Q-smart DPSS, the SolarLaser LQ929 high power pulsed Nd:YAG laser, or the Litron High EnergyPulsed Nd:YAG laser. All of these lasers can produce deep ultravioletradiation within the mJ regime so suitable for ionisation and with shortpulse durations.

It is also possible to passively mode-lock vertical external-cavitysurface-emitting lasers (VECSELs); these are interesting particularlybecause they can deliver a combination of short pulse durations, highpulse repetition rates, and sometimes high average output power, whereasthey are not suitable for high pulse energies.

Post Ionisiation in Two-Pulse Laser Ablation Based Sampling Systems andApparatus

FIG. 17 shows an embodiment where three laser pulses are used to provideablation and post-ionization. Here the first two pulses 40 and 70 areused to ablate material on the small scale similar to the embodiment ofFIG. 13. The third pulse 110 is sent to overlap with the plume ofablated material. It is used to provide further ionization for thismaterial. This step could be necessary to suppress effects ofneutralization that can occur in the ablated plume. The ions are thenextracted through the opening (not shown) in the objective 60.

Post-ionisation requires a high energy density from the laser radiationin a small volume, e.g. 5 μm³ or less. Because the post ionizationvolume is so small it sets the limit on the amount of material that canbe ionized in one go. If a large amount of positive and negative chargesis created in a small volume the motion of the ions formed will bedominated by the local fields resulting from the space charge induced bythe ions and electrons. If there are too many charged ions in a smallvolume, external fields, such as the fields from ion optics present inmass spectrometers used to direct the resulting ions to the detector fordetection, will not be effective at separating positive and negativecharges and such ion clouds will eventually neutralise reducingionisation efficiency. For example, an ion cloud on a scale of 10 μm (indiameter) containing 10000 elemental charges creates an electrostaticpotential that is about 3 V. Since a few eV is the energy holding theelectrons to the atoms it is also the likely energy level of freeelectrons after ionisation. As a result, the ion density on the scale of10000 ions in a volume on the 10 micrometer scale is near the limitwhere the space charge behaviour starts to dominate.

Accordingly, such effects can be avoided by ensuring that the amount ofablated material is kept reasonably small. For example, ablation ofmaterial on the scale of 10×10×10 nm cube to 30×30×30 nm cube or asimilar volume represents the highest amount of material that can betransferred into the post ionization area of a few micrometers in sizewithout creating a strong space change and ion neutralization. Since thesystem can only process 30×30×30 nm cube per single event this createsan opportunity to conduct the imaging with the spatial resolution of 30nm or even 10 nm, to which ion beams can be focused (as discussedherein).

The post ionization laser beam/radiation is co-aligned with the beam ofthe second laser source to within a micrometer precision, as is commonlyobtained in optical setups.

In some embodiments, the post-ionization laser beam/radiation isdirected at an angle to the sample and into the previously ablated areaof the specimen (the arrangement in FIG. 6 for a charged particle-basedsampling and ionization system). This configuration minimizes theinteraction between the laser radiation for post-ionization and theunablated specimen. The laser beam can be focused to a tight focus thatoverlaps with the volume of ablation plume. The laser beam focusing canbe done at high numerical aperture (NA) to facilitate sharp focusing inthe overlapping region and enable rapid laser energy spreading outsideof the overlapping region to minimize the possibility of damage to thesample in the regions surrounding that being sampled.

In some embodiments, numerical aperture of the post-ionization laserbeam/radiation may be constrained in one of the planes. Such anarrangement results in an elliptical focal spot that is extended in theplane of low NA. The elliptical focal spot may improve the degree ofoverlap with the sputtered/ablated plume. Accordingly, in someembodiments, the laser of the post-ionisation system has an ellipticalfocal spot.

In addition to spatial co-ordination, plume generation by the combinedaction of the first and second laser sources described above needs to besynchronised with the delivery of laser radiation to ionise the ablatedmaterial. The speed at which the ablated material may leave the targeton the scale of the speed of sound i.e. 1000 m/s. Thus, to ensurealignment of the ablated plume and the post-ionization laserradiation/beam with 1 micrometer precision requires timing precision onthe scale of 1 ns. In certain aspects, the velocity may be between 1km/s and 10 km/s, such as between 2 km/s and 5 km/s, depending on thetemperature and composition of ablated material. The ablation may beinto atmospheric pressure, partial vacuum pressure, or vacuum pressureas described herein.

Thus, in this mode of operation, the first and second laser sources acttogether to ablate material from the sample, and very shortly afterthat, the ejected material is post-ionised by a pulse of laser radiationfrom a third laser source.

The nano-plasma generated by the action of the first and second laserscontains elemental ions, but where post-ionization is used, these ionsare not extracted directly. Rather, the ejected plume is allowed toexpand, during which time at least some charge neutralization occurs(because the plasma at that point is under high pressure and so dense,meaning that collisional cooling and charge reduction quite rapidlyoccurs. As noted above, the speed of expansion of the nanoplasma plumeis around 1000-5000 m/s. Thus, after a few picoseconds, the plasmagenerated from ablation of e.g. a 10 nm diameter spot on the sample willhave expanded by an order of magnitude, e.g. to a 100 nm³ volume.Additional or re-ionization of the plume after a 10-100 fold dimensionalexpansion of the original nanoplasma volume means that the components ofthe original plume are now significantly more spread out, such that whenionized to reinstate a micrometer scale plasma, collisions are much lesslikely (and thus less likely charge neutralisation) and thus a higherproportion of elemental ions can be extracted from the plume. Withhigher efficiency extraction of ions, including elemental ions derivedfrom labelling atoms, greater sensitivity is achieved.

Accordingly, in some embodiments, two-pulse laser based sampling andablation systems and apparatus comprises a third laser source,configured to ionize plumes of sample material ablated from the sample.

Thus the invention provides a two-pulse laser sampling and ionisationsystem for analysing a sample, such as a biological sample, comprising:

-   -   a sample stage;    -   a first laser source, configured to seed electrons in a sample,        and first focusing optics configured to direct a laser beam        emitted by the first laser source towards the sample stage;    -   a second laser source, configured to ablate sample material        pre-seeded with electrons by the first laser source, and second        focusing optics configured to direct a laser beam emitted by the        second laser source towards sample stage;    -   a third laser source, configured to ionize plumes of sample        material ablated from the sample, and third focusing optics        configured to direct a laser beam emitted by the third laser        source to the volume in which ablated sample material forms a        plume;    -   wherein the second focusing optics is configured to synchronise        a second pulse of laser radiation, from the second laser source,        to arrive at a location on the sample stage directly after the        first pulse of laser radiation, from the first laser source; and    -   wherein the third focusing optics is configured to synchronise a        third pulse of laser radiation to arrive at a volume above the        location on the sample stage directly after the second pulse of        laser radiation.

The third laser provide a fast pulse, an may be less than 30 ps, lessthan 10 ps, less than 1 ps, or less than 500 fs or less than 100 fs,such as at or between 1 fs and 10 ps, at or between 1 fs and 1 ps, at orbetween 1 fs and 500 ps, at or between 100 fs and 500 fs. In someembodiments, the first focusing optics configured to direct a laser beamemitted by the first laser source towards the sample stage is configuredto direct a laser beam emitted by the first laser source to a locationon the sample stage, and the second focusing optics configured to directa laser beam emitted by the second laser source towards the sample stageis configured to direct a laser beam emitted by second first lasersource to the location on the sample stage.

The discussion above of the volume to which the laser radiation from thethird laser source refers to volume above the sample. This is the volumein which an ablation plume forms when material is ejected from thesample. Above is used in this scenario as a term relative to the sample,were the sample in a horizontal plane. If the sample were held in thevertical plane such that the plume following ablation were ejected inthe horizontal axis, the volume “above the sample” to which the laserradiation from the third laser source would be lateral to the sample. Insome embodiments the volume is 5 μm³ or smaller, such as 2.5 μm³ orsmaller, 2 μm³ or smaller, 1 μm³ or smaller, or 100 nm³ or smaller. Insome embodiments the volume is 5 μm³ or smaller, such as 2.5 μm³ orsmaller, 2 μm³ or smaller, 1 μm³ or smaller, or 100 nm³ or smaller. Insome embodiments, the volume extends less than 2 μm, such as less than 1μm, or less than 100 nm from the surface of the sample.

As noted above, the first and second laser sources may representdiscrete lasers or may be derived from the same single laser. Likewise,the third laser source may be a discrete laser or may be derived from(i) the same single laser as the first laser source; (ii) the samesingle laser as the first laser source or (iii) the first, second andthird laser sources may all be derived from the same single laser.

Where different lasers are used, techniques routine in the art, such asprogrammed module comprising instructions can be used to co-ordinatedelivering of the first and second pulses to the sample, such thatpre-seeding and ablation of the pre-seeded area occur in line with thediscussions above, and the pulse from the third laser source to thevolume above the surface of the sample at the ablated location.

In some embodiments, the first and second pulses are synchronised suchthat the time elapsed from the start of the pulse from the first lasersource to the end of the pulse from the second laser source has aduration less than 50 ps, such as shorter than 25 ps, shorter than 10ps, such as shorter than 5 ps, shorter than 2 ps, or shorter than 1 ps,and the pulse from the third laser source arrives at the volume abovethe surface of the sample at the ablated location shorter than 100 nsafter the end of the pulse from the second laser source, In someembodiments, the first and second pulses are synchronised such that thetime elapsed from the start of the pulse from the first laser source tothe end of the pulse from the second laser source has a duration lessthan 50 ps, such as shorter than 25 ps, shorter than 10 ps, such asshorter than 5 ps, shorter than 2 ps, or shorter than 1 ps, and thepulse from the third laser source arrives at the volume above thesurface of the sample at the ablated location shorter than 10 ns afterthe end of the pulse from the second laser source, In some embodiments,the first and second pulses are synchronised such that the time elapsedfrom the start of the pulse from the first laser source to the end ofthe pulse from the second laser source has a duration less than 50 ps,such as shorter than 25 ps, shorter than 10 ps, such as shorter than 5ps, shorter than 2 ps, or shorter than 1 ps, and the pulse from thethird laser source arrives at the volume above the surface of the sampleat the ablated location shorter than 1 ns after the end of the pulsefrom the second laser source,

In some embodiments, the first and second pulses are synchronised suchthat the time elapsed from the start of the pulse from the first lasersource to the end of the pulse from the second laser source has aduration less than 50 ps, such as shorter than 25 ps, shorter than 10ps, such as shorter than 5 ps, shorter than 2 ps, or shorter than 1 ps,and the pulse from the third laser source arrives at the volume abovethe surface of the sample at the ablated location shorter than 100 psafter the end of the pulse from the second laser source, In someembodiments, the first and second pulses are synchronised such that thetime elapsed from the start of the pulse from the first laser source tothe end of the pulse from the second laser source has a duration lessthan 50 ps, such as shorter than 25 ps, shorter than 10 ps, such asshorter than 5 ps, shorter than 2 ps, or shorter than 1 ps, and thepulse from the third laser source arrives at the volume above thesurface of the sample at the ablated location shorter than 50 ps afterthe end of the pulse from the second laser source. In some embodiments,the first and second pulses are synchronised such that the time elapsedfrom the start of the pulse from the first laser source to the end ofthe pulse from the second laser source has a duration less than 50 ps,such as shorter than 25 ps, shorter than 10 ps, such as shorter than 5ps, shorter than 2 ps, or shorter than 1 ps, and the pulse from thethird laser source arrives at the volume above the surface of the sampleat the ablated location shorter than 30 ps after the end of the pulsefrom the second laser source. In some embodiments, the first and secondpulses are synchronised such that the time elapsed from the start of thepulse from the first laser source to the end of the pulse from thesecond laser source has a duration less than 50 ps, such as shorter than25 ps, shorter than 10 ps, such as shorter than 5 μs, shorter than 2 ps,or shorter than 1 ps, and the pulse from the third laser source arrivesa t the volume above the surface of the sample at the ablated locationshorter than 20 ps after the end of the pulse from the second lasersource. In some embodiments, the first and second pulses aresynchronised such that the time elapsed from the start of the pulse fromthe first laser source to the end of the pulse from the second lasersource has a duration less than 50 ps, such as shorter than 25 ps,shorter than 10 ps, such as shorter than 5 ps, shorter than 2 ps, orshorter than 1 ps, and the pulse from the third laser source arrives atthe volume above the surface of the sample at the ablated locationshorter than 10 ps after the end of the pulse from the second lasersource.

Thus the invention provides a two-pulse laser sampling and ionisationsystem for analysing a sample, such as a biological sample, comprising:

-   -   a sample stage;    -   a first laser source, configured to seed electrons in a sample,        and first focusing optics configured to direct a laser beam        emitted by the first laser source towards the sample stage;    -   a second laser source, configured to ablate sample material        pre-seeded with electrons by the first laser source, and second        focusing optics configured to direct a laser beam emitted by the        second laser source towards sample stage;    -   a third laser source, configured to ionize plumes of sample        material ablated from the sample, and third focusing optics        configured to direct a laser beam emitted by the third laser        source to the volume in which ablated sample material forms a        plume;    -   wherein the first laser source, the second laser source and the        third laser source are derived from a single laser, further        comprising at least one beam splitter, at least one two        harmonics generators, and at least one two optical delay lines,    -   wherein one of the harmonics generators is adapted to produce        UV, VUV, EUV or XUV laser radiation (as discussed above for the        first laser source) from the single laser as the first laser        source, and another of the harmonics generators is adapted to        produce IR or visible wavelength laser radiation (as discussed        above for the second laser source) from the single laser as the        second laser source and the third laser source,    -   wherein the second focusing optics comprises an optical delay        line configured to synchronise a second pulse of laser radiation        to arrive at a location on the sample stage such that the end of        the second pulse is shorter than 50 ps following the start of        the first pulse, such as shorter than 25 ps, shorter than 10 ps,        shorter than 5 ps, shorter than 2 ps, or shorter than 1 ps; and    -   wherein the third focusing optics comprises another optical        delay line configured to synchronise a third pulse of laser        radiation to arrive at a volume above the location on the sample        stage shorter than 100 ps after the end of the pulse from the        second laser source, such as shorter than 50 ps, shorter than 30        ps or shorter than 10 ps.

Equipment known in the art can be used to introduce delay between laserpulses. Accordingly, in some embodiments, the apparatus comprises anoptical delay line to introduce delay between laser pulses. Examples ofoptical delay line suitable for use in the present invention are any ofthe optical delay lines commercially available from ThorLabs.

Sample Chamber

The sample chamber of the two-pulse based sampling system may havefeatures in common with the sample chamber of the laser ablation-basedsampling system discussed above. It comprises a stage to support thesample. The stage may be a translation stage, movable in the x-y orx-y-z axes. The sample chamber will also comprise an outlet, throughwhich material removed from the sample by the laser radiation can bedirected. The outlet is connected to the detector, enabling analysis ofthe sample ions.

In some instances, the sample chamber is held under 133322-13.3 Pa, suchas 1333.22-133.322 Pa. In some instances, the sample chamber is heldunder a vacuum. Accordingly, in some instances, the sample chamberpressure is lower than 50000 Pa, lower than 10000 Pa, lower than 5000Pa, lower than 1000 Pa, lower than 500 Pa, lower than 100 Pa, lower than10 Pa, lower than 1 Pa, around 0.1 Pa or less than 0.1 Pa, such as 0.01Pa or lower. For instance, partial vacuum pressure may be around 200-700Pa, and vacuum pressure 0.2 Pa or lower. Typical gases such as Argon,Helium, Nitrogen and mixtures thereof.

The selection of whether the sample pressure is at atmospheric pressureunder a (partial) vacuum depends on the particular analysis beingperformed, as will be understood by one of skill in the art. Forinstance, at atmospheric pressure, sample handing is easier, and softerionisation may be applied. Further, the presence of gas molecules may bedesired so as to enable the phenomenon of collisional cooling to occur,which can be of interest when the label is a large molecule, thefragmentation of which is not desired, e.g. a molecular fragmentcomprising a labelling atom or combination thereof. Alternatively, ininstances where laser radiation is used to post-ionise material thepresence of gas molecules and collisional cooling may be advantageous toallow the cooling of the nanoplasma generated at the surface of thesample (i.e. following charged particle bombardment and laserillumination of the sample to generate the energy pumping state) andexpansion of the plume of ablated material and before re-ionization in apost-ionization system.

The selection of whether the sample pressure is at atmospheric pressureunder a (partial) vacuum depends on the particular analysis beingperformed, as will be understood by one of skill in the art. Forinstance, at atmospheric pressure, sample handing is easier, and softerionisation may be applied. Further, the presence of gas molecules may bedesired so as to enable the phenomenon of collisional cooling to occur,which can be of interest when the label is a large molecule, thefragmentation of which is not desired, e.g. a molecular fragmentcomprising a labelling atom or combination thereof.

Holding the sample chamber under vacuum can prevent collisions betweensample ions generated and other particles within the chamber.

Ion Microscope and Optics

The sample ions are captured from the sample via an electrostatic lenspositioned near to the sample, known in the art as an immersion lens (oran extraction lens). The immersion lens removes the secondary ionsimmediately from the locality of the sample. This is typically achievedby the sample and the lens having a large difference in voltagepotential. Depending on the polarity of the sample vis-à-vis theimmersion lens, positive or negative secondary ions are captured by theimmersion lens. The polarity of the sample ions as captured by theimmersion lens is independent of the polarity of the ions of the chargedparticle beam.

The sample ions are then transferred to the detector by via one or morefurther electrostatic lenses (known as transfer lenses in the art). Thetransfer lens(es) focus(es) the beam of secondary ions into thedetector. Typically, in systems with multiple transfer lenses, only onetransfer lens is engaged in a given analysis. Each lens may provide adifferent magnification of the sample surface. Commonly, further ionmanipulation components are present between the immersion lens and thedetector, for example one or more apertures, mass filters or sets ofdeflector plates. Together, the immersion lens, transfer lens, and anyfurther components, form the ion microscope. Components for theproduction of an ion microscope are available from commercial supplierse.g. Agilent.

Camera

The system may also comprise a camera. Camera systems are discussedabove in relation to laser ablation sampling systems, and the featuresof the above camera can also be present in the secondary ion generationsystem, except where incompatible (e.g. it can be connected to a lightmicroscope, such as a confocal microscope, but it is not possible tofocus a primary ion beam through the same optics as the light which isdirected to the camera, because one beam is ions and the other photons).

Methods of Using a Two-Pulse Laser Based Apparatus

The invention also provides methods of analysing a biological sampleusing an apparatus or sampling and ionisation system as described inthis section. Accordingly, the features discussed above with respect tothe apparatus are suitable features for incorporation in the methodclaims. Accordingly, the invention provides a method for analysing asample comprising the steps of:

-   -   (i) seeding electrons in a location on a sample using laser        radiation emitted by a first laser source; and    -   (ii) ablating sample material, from the location on the sample        pre-seeded with electrons by the first laser source, using laser        radiation emitted by a second laser source.

Step (ii) may also ionize (e.g., sustainably ionize) the seeded sample.In some embodiments, step (ii) is completed within shorter than 50 psfollowing the start of the first pulse, such as shorter than 25 ps,shorter than 10 ps, such as shorter than 5 ps, shorter than 2 ps, orshorter than 1 ps. of the commencement of step (i). In some embodiments,step (i) is performed with a pulse of laser radiation of 5 ps orshorter, such as 2 ps or shorter, 1 ps or shorter, 500 fs or shorter,400 fs or shorter, 300 fs or shorter, 200 fs or shorter or 100 fs orshorter, 50 fs or shorter, 40 fs or shorter, 30 fs or shorter, 20 fs orshorter or 10 fs or shorter. In some embodiments, the laser radiationemitted by the first lase-r source is 200 nm or shorter, 175 nm orshorter, 150 nm or shorter, 125 nm or shorter, 100 nm or shorter, 75 nmor shorter, 50 nm or shorter, 25 nm or shorter, 20 nm or shorter or 15nm. In some embodiments, the laser radiation emitted by the first lasersource is between 10-200 nm, between 10-175 nm, between 10-150 nm,between 10-125 nm, between 10-100 nm, between 10-75 nm, between 10-50nm, between 10-25 nm, between 10-20 nm, or between 10-15 nm. In someembodiments, the pulse energy of the first laser source is in the picoJto femtoJ range, such as between 1 picoJ-999 femtoJ, between 10picoJ-100 femtoJ, or between 100 picoJ-1 femtoJ. In some embodiments,the diameter of the location is 100 nm or shorter, such as 75 nm orshorter, 50 nm or shorter or around 30 nm.

In some embodiments, step (ii) is performed with a pulse of laserradiation of 5 ps or shorter, such as 2 ps or shorter, 1 ps or shorter,500 fs or shorter, 400 fs or shorter, 300 fs or shorter, 200 fs orshorter or 100 fs or shorter, 50 fs or shorter, 40 fs or shorter, 30 fsor shorter, 20 fs or shorter or 10 fs or shorter. In some embodiments,the wavelength of laser radiation emitted by the second laser source isbetween 400 nm-100 μm, such as between 400 nm-10 μm, between 400 nm-1μm, between 400 nm-900 nm, between 400 nm-800 nm, between 400 nm-700 nm,between 400 nm-600 nm or between 500 nm-600 nm, between 200 nm-100 μm,between 200 nm-10 μm, or between 200 nm-1 μm. In some embodiments, thepulse energy of the second laser source is in the nanoJoule range, suchas between 1 nanoJ-1 μJ, between 10 nanoJ-500 nanoJ, between 50nanoJ-250 nanoJ, or around 100 nanoJ.

In some embodiments, the method further comprises illuminating the plumeof material ablated from the sample in step (ii) with laser radiationfrom a third laser source. In some embodiments, the laser radiation fromthe third laser source arrives at a volume above the location on thesample shorter than 10 ns after the end of the pulse from the secondlaser source, such as shorter than 1 ns, shorter than 100 ps, shorterthan 50 ps, shorter than 30 ps or shorter than 10 ps

Post-Ionisation Systems and Methods

In any of the method of sampling described herein, including by initialradiation from a first energy source (e.g., a laser, ion beam, orelectron beam), material released from the sample may be ionized by alaser at or near the sample surface. The laser used for ionisation maybe an IR or visible laser, and may be on the picosecond scale (e.g.,1-1000 ps, 5-100 ps, 10-50 ps). The material released may be ionized bythe laser within 100 ps to 10 ns of the initial radiation, allowing thematerial to expand past the critical density (before whichneutralization would significantly reduce long-term ion formation). Thismay create a microplasma (above the surface), as oppose to a nanoplasmathat would be formed from direct ionization at the sample surface. Thesample (e.g., chamber housing the sample) may be held at vacuum orpartial vacuum pressure to allow expansion of the ablation plume pastthe critical density and/or improve the ability of ion optics to directresulting ions. Partial vacuum pressure (e.g., 10-10,000 Pa or 100-1,000Pa) may allow for collisional cooling and/or charge reduction, and mayimprove the ion optics.

2. Mass Detector System

Exemplary types of mass detector system include quadrupole, time offlight (TOF), magnetic sector, high resolution, single or multicollectorbased mass spectrometers. A magnetic sector instrument is particularlysuited for a high rate of recording of 1 megapixel per second and above.

The time taken to analyse the ionised material will depend on the typeof mass analyser which is used for detection of ions. For example,instruments which use Faraday cups are generally too slow for analysingrapid signals. Overall, the desired imaging speed, resolution and degreeof multiplexing will dictate the type(s) of mass analyser which shouldbe used (or, conversely, the choice of mass analyser will determine thespeed, resolution and multiplexing which can be achieved).

Mass spectrometry instruments that detect ions at only onemass-to-charge ratio (m/Q, commonly referred to as m/z in MS) at a time,for example using a point ion detector, will give poor results inimaging detecting. Firstly, the time taken to switch betweenmass-to-charge ratios limits the speed at which multiple signals can bedetermined, and secondly, if ions are at low abundance then signals canbe missed when the instrument is focused on other mass-to-charge ratios.Thus it is preferred to use a technique which offers substantiallysimultaneous detection of ions having different m/Q values.

Detector Types

Quadrupole Detector

Quadrupole mass analysers comprise four parallel rods with a detector atone end. An alternating RF potential and fixed DC offset potential isapplied between one pair of rods and the other so that one pair of rods(each of the rods opposite each other) has an opposite alternativepotential to the other pair of rods. The ionised sample is passedthrough the middle of the rods, in a direction parallel to the rods andtowards the detector. The applied potentials affect the trajectory ofthe ions such that only ions of a certain mass-charge ratio will have astable trajectory and so reach the detector. Ions of other mass-chargeratios will collide with the rods.

Magnetic Sector Detector

In magnetic sector mass spectrometry, the ionised sample is passedthrough a curved flight tube towards an ion detector. A magnetic fieldapplied across the flight tube causes the ions to deflect from theirpath. The amount of deflection of each ion is based on the mass tocharge ratio of each ion and so only some of the ions will collide withthe detector—the other ions will be deflected away from the detector. Inmulticollector sector field instruments, an array of detectors is beused to detect ions of different masses. In some instruments, such asthe ThermoScientific Neptune Plus, and Nu Plasma II, the magnetic sectoris combined with an electrostatic sector to provide a double-focussingmagnetic sector instrument that analyses ions by kinetic energy, inaddition to mass to charge ratio. In particular those multidetectorshaving a Mattauch-Herzog geometry can be used (e.g. the SPECTRO MS,which can simultaneously record all elements from lithium to uranium ina single measurement using a semiconductor direct charge detector).These instruments can measure multiple m/Q signals substantiallysimultaneously. Their sensitivity can be increased by including electronmultipliers in the detectors. Array sector instruments are alwaysapplicable, however, because, although they are useful for detectingincreasing signals, they are less useful when signal levels aredecreasing, and so they are not well suited in situations where labelsare present at particularly highly variable concentrations.

Time of Flight (TOF) Detector

A time of flight mass spectrometer comprises a sample inlet, anacceleration chamber with a strong electric field applied across it, andan ion detector. A packet of ionised sample molecules is introducedthrough the sample inlet and into the acceleration chamber. Initially,each of the ionised sample molecules has the same kinetic energy but asthe ionised sample molecules are accelerated through the accelerationchamber, they are separated by their masses, with the lighter ionisedsample molecules travelling faster than heaver ions. The detector thendetects all the ions as they arrive. The time taking for each particleto reach the detector depends on the mass to charge ratio of theparticle.

Thus a TOF detector can quasi-simultaneously register multiple masses ina single sample. In theory TOF techniques are not ideally suited to ICPion sources because of their space charge characteristics, but TOFinstruments can in fact analyse an ICP ion aerosol rapidly enough andsensitively enough to permit feasible single-cell imaging. Whereas TOFmass analyzers are normally unpopular for atomic analysis because of thecompromises required to deal with the effects of space charge in the TOFaccelerator and flight tube, tissue imaging according to the subjectdisclosure can be effective by detecting only the labelling atoms, andso other atoms (e.g. those having an atomic mass below 100) can beremoved. This results in a less dense ion beam, enriched in the massesin (for example) the 100-250 dalton region, which can be manipulated andfocused more efficiently, thereby facilitating TOF detection and takingadvantage of the high spectral scan rate of TOF. Thus rapid imaging canbe achieved by combining TOF detection with choosing labelling atomsthat are uncommon in the sample and ideally having masses above themasses seen in an unlabelled sample e.g. by using the higher masstransition elements. Using a narrower window of label masses thus meansthat TOF detection to be used for efficient imaging.

Suitable TOF instruments are available from Tofwerk, GBC ScientificEquipment (e.g. the Optimass 9500 ICP-TOFMS), and Fluidigm Canada (e.g.the CyTOF™ and CyTOF™2 pinstruments). These CyTOF™ instruments havegreater sensitivity than the Tofwerk and GBC instruments and are knownfor use in mass cytometry because they can rapidly and sensitivelydetect ions in the mass range of rare earth metals (particularly in them/Q range of 100-200; see Bandura et al. (2009; Anal. Chem.,81:6813-22)). Thus these are preferred instruments for use with thedisclosure, and they can be used for imaging with the instrumentsettings already known in the art e.g. Bendal et al. (2011; Science 332,687-696) & Bodenmiller et al. (2012; Nat. Biotechnol. 30:858-867). Theirmass analysers can detect a large number of markers quasi-simultaneouslyat a high mass-spectrum acquisition frequency on the timescale ofhigh-frequency laser ablation or sample desorption. They can measure theabundance of labelling atoms with a detection limit of about 100 percell, permitting sensitive construction of an image of the tissuesample. Because of these features, mass cytometry can now be used tomeet the sensitivity and multiplexing needs for tissue imaging atsubcellular resolution. By combining the mass cytometry instrument witha high-resolution laser ablation sampling system and a rapid-transitlow-dispersion sample chamber it has been possible to permitconstruction of an image of the tissue sample with high multiplexing ona practical timescale.

The TOF may be coupled with a mass-assignment corrector. The vastmajority of ionisation events generate M⁺ ions, where a single electronhas been knocked out of the atom. Because of the mode of operation ofthe TOF MS there is sometimes some bleeding (or cross-talk) of the ionsof one mass (M) into the channels for neighbouring masses (M±1), inparticular where a large number of ions of mass M are entering thedetector (i.e. ion counts which are high, but not so high that an iondeflector positioned between the sampling ionisation system and MS wouldprevent them from entering the MS, if the apparatus were to comprisesuch an ion deflector). As the arrival time of each M⁺ ion at thedetector follows a probability distribution about a mean (which is knownfor each M), when the number of ions at mass M⁺ is high, then some willarrive at times that would normally be associated with the M-1⁺ or M+1⁺ions. However, as each ion has a known distribution curve upon enteringthe TOF MS, based on the peak in the mass M channel it is possible todetermine, the overlap of ions of mass M into the M±1 channels (bycomparison to the known peak shape). The calculation is particularlyapplicable for TOF MS, because the peak of ions detected in a TOF MS isasymmetrical. Accordingly it is therefore possible to correct thereadings for the M-1, M and M+1 channels to appropriately assign all ofthe detected ions to the M channel. Such corrections have particular usein correcting imaging data due to the nature of the large packets ofions produced by sampling and ionisation systems such as those disclosedherein involving laser ablation (or desorption as discussed below) asthe techniques for removing material from the sample. Programs andmethods for improving the quality of data by de-convoluting the datafrom TOF MS are discussed in WO2011/098834, U.S. Pat. No. 8,723,108 andWO2014/091243.

Dead-Time Corrector

As noted above, signals in the MS are detected on the basis ofcollisions between ions and the detector, and the release of electronsfrom the surface of the detector hit by the ions. When a high count ofions is detected by the MS resulting in the release of a large number ofelectrons, the detector of the MS can become temporarily fatigued, withthe result that the analog signal output from the detector istemporarily depressed for one or more of the subsequent packets of ions.In other words, a particularly high count of ions in a packet of ionisedsample material causes a lot of electrons to be released from thedetector surface and secondary multiplier in the process of detectingthe ions from that packet of ionised sample material, meaning that fewerelectrons are available to be released when the ions in subsequentpackets of ionised sample material hit the detector, until the electronsin the detector surface and secondary amplifier are replenished.

Based on a characterisation of the behaviour of the detector, it ispossible to compensate for this dead-time phenomenon. A first step is toanalyse the ion peak in the analog signal resulting from the detectionof the nth packet of ionised sample material by the detector. Themagnitude of the peak may be determined by the height of the peak, bythe area of the peak, or by a combination of peak height and peak area.

The magnitude of the peak is then compared to see if it exceeds apredetermined threshold. If the magnitude is below this threshold, thenno correction is necessary. If the magnitude is above the threshold,then correction of the digital signal from at least one subsequentpacket of ionised sample material will be performed (at least the(n+1)th packet of ionised sample material, but possibly further packetsof ionised sample material, such as (n+2)th, (n+3)th, (n+4)th etc.) tocompensate for the temporary depression of the analog signal from thesepackets of ionised sample material resulting from the fatiguing of thedetector caused by the nth packet of ionised sample material. Thegreater the magnitude of the peak of the nth packet of ionised samplematerial, the more peaks from subsequent packets of ionised samplematerial will need to be corrected and the magnitude of correction willneed to be greater. Methods for correcting such phenomena are discussedin Stephan et al. (1994; Vac. Sci. Technol. 12:405), Tyler and Peterson(2013; Surf Interface Anal. 45:475-478), Tyler (2014; Surf InterfaceAnal. 46:581-590), WO2006/090138 and U.S. Pat. No. 6,229,142, and thesemethods can be applied by the dead-time corrector to the data, asdescribed herein.

Analyser Apparatus Based on Optical Emission Spectra Detection

1. Sampling and Ionisation Systems

a. Laser Ablation Based Sampling and Ionising System

The laser ablation sampling system comprising a laser scanning systemdescribed above in relation to mass-based analysers can be employed inan OES detector-based system. For detection of atomic emission spectra,most preferably, an ICP is used to ionise the sample material removedfrom the sample, but any hard ionisation technique that can produceelemental ions can be used.

As appreciated by one of skill in the art, certain optional furthercomponents of the laser ablation based sampling and ionising systemabove, described in relation to avoiding overload of the mass-baseddetector, may not be applicable to all OES detector-based systems, andwould not be incorporated, if inappropriate, by the skilled artisan.

2. Photodetectors Exemplary types of photodetectors includephotomultipliers and charged-coupled devices (CCDs). Photodetetors maybe used to image the sample and/or identify a feature/region of interestprior to imaging by elemental mass spectrometry.

Photomultipliers comprise a vacuum chamber comprising a photocathode,several dynodes, and an anode. A photon incident on the photocathodecauses the photocathode to emit an electron as a consequence of thephotoelectric effect. The electron is multiplied by the dynodes due tothe process of secondary emission to produce a multiplied electroncurrent, and then the multiplied electron current is detected by theanode to provide a measure of detection of electromagnetic radiationincident on the photocathode. Photomultipliers are available from, forexample, ThorLabs.

A CCD comprises a silicon chip containing an array of light-sensitivepixels. During exposure to light, each pixel generates an electriccharge in proportion to the intensity of light incident on the pixel.After the exposure, a control circuit causes a sequence of transfers ofelectric charge to produce a sequence of voltages. These voltages canthen be analysed to produce an image. Suitable CCDs are available from,for example, Cell Biosciences.

Constructing an Image

The apparatus above can provide signals for multiple atoms in packets ofionised sample material removed from the sample. Detection of an atom ina packet of sample material reveals its presence at the position ofablation, be that because the atom is naturally present in the sample orbecause the atom has been localised to that location by a labellingreagent. By generating a series of packets of ionised sample materialfrom known spatial locations on the sample's surface the detectorsignals reveal the location of the atoms on the sample, and so thesignals can be used to construct an image of the sample. By labellingmultiple targets with distinguishable labels it is possible to associatethe location of labelling atoms with the location of cognate targets, sothe method can build complex images, reaching levels of multiplexingwhich far exceed those achievable using traditional techniques such asfluorescence microscopy.

Assembly of signals into an image will use a computer and can beachieved using known techniques and software packages. For instance, theGRAPHIS package from Kylebank Software may be used, or other packagessuch as TERAPLOT can also be used. Imaging using MS data from techniquessuch as MALDI-MSI is known in the art e.g. Robichaud et al. (2013; J AmSoc Mass Spectrom 24 5:718-21) discloses the ‘MSiReader’ interface toview and analyze MS imaging files on a Matlab platform, and Klinkert etal. (2014; Int J Mass Spectromhttp://dx.doi.org/10.1016/j.ijms.2013.12.012) discloses two softwareinstruments for rapid data exploration and visualization of both 2D and3D MSI data sets in full spatial and spectral resolution e.g. the‘Datacube Explorer’ program. In addition, systems and methods describedherein may be run according to software. For example,

Images obtained using the methods disclosed herein can be furtheranalysed e.g. in the same way that IHC results are analysed. Forinstance, the images can be used for delineating cell sub-populationswithin a sample, and can provide information useful for clinicaldiagnosis. Similarly, SPADE analysis can be used to extract a cellularhierarchy from the high-dimensional cytometry data which methods of thedisclosure provide (Qiu et al. (2011; Nat. Biotechnol. 29:886-91)).

Samples

Certain aspects of the disclosure provide a method of imaging abiological sample. Such samples can comprise a plurality of cells whichcan be subjected to imaging mass cytometry (IMC) in order to provide animage of these cells in the sample. In general, the invention can beused to analyse tissue samples which are now studied byimmunohistochemistry (IHC) techniques, but with the use of labellingatoms which are suitable for detection by mass spectrometry (MS) oroptical emission spectrometry (OES). Furthermore, the present inventionprovides various techniques for preparing tissue samples in order toprovide improved resolution over IMC and IMS techniques using samplesprepared in a traditional manner. In particular, the present inventionprovides techniques for preparing samples which are suitable for imagingby electron microscopy, for preparing ultrathin samples, and acombination thereof. These methods are described further herein.

Any suitable tissue sample can be used in the methods described herein.For example, the tissue can include tissue from one or more ofepithelium, muscle, nerve, skin, intestine, pancreas, kidney, brain,liver, blood (e.g. a blood smear), bone marrow, buccal swipes, cervicalswipes, or any other tissue. The biological sample may be animmortalized cell line or primary cells obtained from a living subject.For diagnostic, prognostic or experimental (e.g., drug development)purposes the tissue can be from a tumor. In some embodiments, a samplemay be from a known tissue, but it might be unknown whether the samplecontains tumor cells. Imaging can reveal the presence of targets whichindicate the presence of a tumor, thus facilitating diagnosis. Tissuefrom a tumor may comprise immune cells that are also characterized bythe subject methods, and may provide insight into the tumor biology. Thetissue sample may comprise formalin-fixed, paraffin-embedded (FFPE)tissue. The tissues can be obtained from any living multicellularorganism, such as a mammal, an animal research model (e.g., of aparticular disease, such as an immunodeficient rodent with a human tumorxenograft), or a human patient.

The tissue sample may be a section e.g. having a thickness within therange of 2-10 μm, such as between 4-6 μm. Techniques for preparing suchsections are well known from the field of IHC e.g. using microtomes,including dehydration steps, fixation, embedding, permeabilization,sectioning etc. Thus, a tissue may be chemically fixed and then sectionscan be prepared in the desired plane. Cryosectioning or laser capturemicrodissection can also be used for preparing tissue samples. Samplesmay be permeabilised e.g. to permit uptake of reagents for labelling ofintracellular targets (see above).

The size of a tissue sample to be analysed will be similar to currentIHC methods, although the maximum size will be dictated by the laserablation apparatus, and in particular by the size of sample which canfit into its sample chamber. A size of up to 5 mm×5 mm is typical, butsmaller samples (e.g. 1 mm×1 mm) are also useful (these dimensions referto the size of the section, not its thickness).

In addition to being useful for imaging tissue samples, the disclosurecan instead be used for imaging of cellular samples such as monolayersof adherent cells or of cells which are immobilised on a solid surface(as in conventional immunocytochemistry). These embodiments areparticularly useful for the analysis of adherent cells that cannot beeasily solubilized for cell-suspension mass cytometry. Thus, as well asbeing useful for enhancing current immunohistochemical analysis, thedisclosure can be used to enhance immunocytochemistry.

Ultrathin Samples

As discussed above, traditional IMC and IMS techniques use tissuesamples that are several micrometres thick. However, some of theembodiments of the invention described herein, for example the apparatusfor analysing a biological sample comprising an immersion mediumpositioned between the objective lens and the sample stage (see page 9above), are not suitable for use with samples of such a thicknessbecause the ablation region is typically of the order of 100 nm in allthree dimensions, or at least in the lateral dimensions.

Therefore, the present invention provides a method of preparing abiological sample for analysis comprising labelling the sample withlabelling atoms (labelling atoms are described further herein) andsectioning the sample into thin sections, optionally wherein the sampleis sectioned into sections of thickness of less than 10 micrometers orbelow, such as 1 micrometer or below, or 100 nm or below, or 50 nm orbelow, or 30 nm or below. The invention also provides a method ofpreparing a biological sample for analysis comprising sectioning thesample into thin sections and labelling the sample with labelling atoms(labelling atoms are described further herein), optionally wherein thesample is sectioned into sections of thickness of less than 10micrometers or below, such as 1 micrometer or below, or 100 nm or below,or 50 nm or below, or 30 nm or below. An automated microtome, such asthe ATUMtome available from RM Boeckeler, can be used to section thesample into sections of a thickness in accordance with the method of thepresent invention.

Samples prepared according to the method set out above can be used withany of the IMC and IMS techniques described herein. However, samplesprepared according to the method set out above are particularly suitedto analysis by any of the apparatus comprising an immersion mediumpositioned between the objective lens and the sample stage describedherein.

Furthermore, samples prepared according to the method set out above arealso suited to analysis by any one of the sputtering based sampling andionising systems set out above.

Sample Carrier

In certain embodiments, the sample may be immobilized on a solid support(i.e. a sample carrier), to position it for imaging mass spectrometry.The solid support may be optically transparent, for example made ofglass or plastic. Where the sample carrier is optically transparent, itenables ablation of the sample material through the carrier, asillustrated in FIGS. 3-5 and 8-10. Ablation through the material of thesample carrier has particular advantages when laser radiation isdirected onto the sample through an immersion medium.

Sometimes, the sample carrier will comprise features that act asreference points for use with the apparatus and methods describedherein, for instance to allow the calculation of the relative positionof features/regions of interest that are to be ablated or desorbed andanalysed. The reference points may be optically resolvable, or may beresolvable by mass analysis.

Target Elements

In imaging mass spectrometry, the distribution of one or more targetelements (i.e., elements or elemental isotopes) may be of interest. Incertain aspects, target elements are labelling atoms as describedherein. A labelling atom may be directly added to the sample alone orcovalently bound to or within a biologically active molecule. In certainembodiments, labelling atoms (e.g., metal tags) may be conjugated to amember of a specific binding pair (SBP), such as an antibody (that bindsto its cognate antigen), aptamer or oligonucleotide for hybridizing to aDNA or RNA target, as described in more detail below. Labelling atomsmay be attached to an SBP by any method known in the art. In certainaspects, the labelling atoms are a metal element, such as a lanthanideor transition element or another metal tag as described herein. Themetal element may have a mass greater than 60 amu, greater than 80 amu,greater than 100 amu, or greater than 120 amu. Mass spectrometersdescribed herein may deplete elemental ions below the masses of themetal elements, so that abundant lighter elements do not createspace-charge effects and/or overwhelm the mass detector.

Labelling of the Tissue Sample

The disclosure produces images of samples which have been labelled withlabelling atoms, for example a plurality of different labelling atoms,wherein the labelling atoms are detected by an apparatus capable ofsampling specific, preferably subcellular, areas of a sample (thelabelling atoms therefore represent an elemental tag). The reference toa plurality of different atoms means that more than one atomic speciesis used to label the sample. These atomic species can be distinguishedusing a mass detector (e.g. they have different m/Q ratios), such thatthe presence of two different labelling atoms within a plume gives riseto two different MS signals. The atomic species can also bedistinguished using an optical spectrometer (e.g. different atoms havedifferent emission spectra), such that the presence of two differentlabelling atoms within a plume gives rise to two different emissionspectral signals.

Labelling for Electron Microscopy (EM) Techniques

Immunoelectron microscopy is the use of electron microscopy to study theultrastructure of tissues or cells. In immunoelectron microscopy,electron microscopy is used to detect the antibody SPBs labelled withheavy metal particles, such as those described further herein. Scanningelectron microscopy (SEM) and transmission electron microscopy (TEM) areboth well known electron microscopy techniques and can be used to detectthe antibody SPBs. Scanning electron microscopy (SEM) is used to detectsignals resulting from the interaction of electrons with the surface ofthe sample. Transmission electron microscopy (TEM) is used to detectimages produced by electrons passing through ultrathin specimens (lessthan 50 nm). Standard protocol for preparing samples for immunoelectronmicroscopy involves tissue fixation, inclusion in an appropriateembedding resin and subsequent ultrathin sectioning (to sections ofthickness of 50 nm or less) and incubation with specific antibodies forthe molecules whose ultrastructural location needs to be determined.

The present invention utilises techniques for preparing samples that aresuitable for analysis by electron microscopy. In one aspect, the presentinvention provides a method for preparing a biological sample foranalysis comprising staining the sample with a contrast agent forelectron microscopy (EM); and labelling the sample with labelling atoms.In some embodiments, the method comprises prior to staining orlabelling, the step of preparing an ultrathin section of the biologicalsample, such as 100 nm or thinner, 50 nm or thinner or 30 nm or thinner.In this way, the present invention provides samples which are suitablefor imaging using EM prior to analysis using any of the IMC and IMStechniques discussed herein. Thus, the present invention providesopportunities to provide imaging of a higher resolution and/ormultiplexity than traditional IMC and IMS techniques.

In some embodiments of the invention, the contrast agent for EM isvisible in an imaging mass cytometer or imaging mass spectrometer.Accordingly, the present invention provides a method of preparing abiological sample in which the contrast agent includes at least one ofosmium tetroxide, gold, silver and iridium. Thus, the biological sampleprepared according to the method of the present invention can be readfirst by EM, thereby providing spatial resolution on a 3 nm scale forone (affinity) channel (the EM contrast agent), and then read by IMC/IMSwith a spatial resolution of 100 nm with as many affinity channels asprovided by the labelling atoms (for example, over 40 affinitychannels). One advantage of utilising contrast agents for EM which arealso visible in a mass cytometer, such as osmium tetroxide or iridium,is that the IMC/IMS can easily read out the contrast agents and so theimages obtained by EM and IMC/IMS can be overlaid and co-registered. TheEM provides fine detail and IMC/IMS provides the advantage of monitoringbiological states of the specimen on many affinity channels. In thisway, preparing a biological sample for analysis according to the presentinvention provides an opportunity to expand IMC capabilities toultrastructural analysis of cells, obtaining higher resolution imagesthan those provided by traditional IMC and IMS.

Furthermore, preparing a biological sample in a style suitable forelectron microscopy generally includes preparing the samples in a resin,as opposed to formalin fixed paraffin-embedded (FFPE; which is discussedabove). FFPE based samples can shift and smear when drying, reducingimage resolution. On the other hand, resin in the specimen facilitatesuniformity of ablation further increases the resolution.

Moreover, preparing a biological in a style suitable for electronmicroscopy generally includes sectioning the sample into thin samples,as described for example on page 40. Accordingly, the present inventionprovides a method of preparing a biological sample for analysiscomprising: first comprising sectioning the sample into thin sections;optionally wherein the sample is sectioned into sections of thickness ofless than 10 micrometers or below, such as 1 micrometer or below, or 100nm or below, or 30 nm or below.

In some embodiments of the invention, the labelling atoms include atleast one of gold and lanthanide labelled antibodies.

Samples prepared according to the method set out above can be used withany of the IMC and IMS techniques described herein. Accordingly, thepresent invention provides a method of analysing a biological samplecomprising:

-   -   directing a beam of radiation emitted by the laser source        towards a location on the sample to produce an ablated plume of        sample material;    -   ionising the ablated plume of sample material; and    -   detecting the sample ions from the sample material;

optionally, wherein the method of the present invention comprises firstperforming electron microscopy. In some embodiments, the ions aredetected by a TOF MS. In some embodiments, the sample material isionised by ICP.

In order to reconstruct the image of a single layer of the thickness ofa biological cell or to read a thicker specimen layer by layer andgenerate a 3D image, as discussed further herein, the sample preferablyhas a thickness of 100 micrometers or below, such as 10 micrometers orbelow, or 100 nm or below, or 50 nm or below, or 30 nm or below. In someembodiments described in more detail herein, the immersion medium isreferred to as an immersion lens. The invention also provides use of abiological sample with a thickness of 100 nm or less in a method ofimaging mass cytometry. The invention also provides use of a biologicalsample labelled with lanthanide and/or actinide atoms in an electronmicroscopy method.

The present invention also provides a method of analysing a biologicalsample comprising performing electron microscopy (such as transmissionelectron microscopy) on the sample, and then performing IMC on thesample. In some embodiments, the invention provides a method ofanalysing a biological sample comprising the steps of:

-   -   a) performing transmission electron microscopy on the sample to        generate an EM image;    -   b) sampling and ionising material from one or more locations on        the sample, comprising the step of directing laser radiation at        each location on the sample to generate a plume of sample        material from each location,    -   c) detecting ions in the plumes of sample material, whereby        detection of the ions in the plumes permits construction of an        element image of the sample.

This method sometimes further comprises the step of overlaying the EMimage and the element image. In some embodiments, the locations areknown locations.

In some embodiments, the invention provides a method of analysing abiological sample comprising the steps of:

-   -   a) staining the biological sample with a contrast reagent for EM        and labelling one or more a target molecules in the tissue        sample with labelling atoms, to provide a stained and labelled        sample;    -   b) performing transmission electron microscopy on the stained        and labelled sample to generate an EM image;    -   c) sampling and ionising material from one or more known        locations on the stained and labelled sample, comprising the        step of directing laser radiation at each known location on the        sample to generate a plume of sample material from each known        location,    -   d) detecting ions in the plumes of sample material, whereby        detection of ions of the labelling atoms in the plumes permits        construction of an element image of the sample.

This method sometimes further comprises the step of overlaying the EMimage and the element image. In some embodiments, the laser radiationsamples material and ionises it. In some embodiments, ionisation togenerate ions for detection is performed separately from sampling by anICP.

Mass Tagged Reagents

Mass-tagged reagents as used herein comprise a number of components. Thefirst is the SBP. The second is the mass tag. The mass tag and the SBPare joined by a linker, formed at least in part of by the conjugation ofthe mass tag and the SBP. The linkage between the SBP and the mass tagmay also comprise a spacer. The mass tag and the SBP can be conjugatedtogether by a range of reaction chemistries. Exemplary conjugationreaction chemistries include thiol maleimide, NHS ester and amine, orclick chemistry reactivities (preferably Cu(I)-free chemistries), suchas strained alkyne and azide, strained alkyne and nitrone and strainedalkene and tetrazine.

Mass Tags

The mass tag used in the present invention can take a number of forms.Typically, the tag comprises at least one labelling atom. A labellingatom is discussed herein below.

Accordingly, in its simplest form, the mass tag may comprise ametal-chelating moiety which is a metal-chelating group with a metallabelling atom co-ordinated in the ligand. In some instances, detectingonly a single metal atom per mass tag may be sufficient. However, inother instances, it may be desirable of each mass tag to contain morethan one labelling atom. This can be achieved in a number of ways, asdiscussed below.

A first means to generate a mass tag that can contain more than onelabelling atom is the use of a polymer comprising metal-chelatingligands attached to more than one subunit of the polymer. The number ofmetal-chelating groups capable of binding at least one metal atom in thepolymer can be between approximately 1 and 10,000, such as 5-100,10-250, 250-5,000, 500-2,500, or 500-1,000. At least one metal atom canbe bound to at least one of the metal-chelating groups. The polymer canhave a degree of polymerization of between approximately 1 and 10,000,such as 5-100, 10-250, 250-5,000, 500-2,500, or 500-1,000. Accordingly,a polymer based mass tag can comprise between approximately 1 and10,000, such as 5-100, 10-250, 250-5,000, 500-2,500, or 500-1,000labelling atoms.

The polymer can be selected from the group consisting of linearpolymers, copolymers, branched polymers, graft copolymers, blockpolymers, star polymers, and hyperbranched polymers. The backbone of thepolymer can be derived from substituted polyacrylamide,polymethacrylate, or polymethacrylamide and can be a substitutedderivative of a homopolymer or copolymer of acrylamides,methacrylamides, acrylate esters, methacrylate esters, acrylic acid ormethacrylic acid. The polymer can be synthesised from the groupconsisting of reversible addition fragmentation polymerization (RAFT),atom transfer radical polymerization (ATRP) and anionic polymerization.The step of providing the polymer can comprise synthesis of the polymerfrom compounds selected from the group consisting of N-alkylacrylamides, N,N-dialkyl acrylamides, N-aryl acrylamides, N-alkylmethacrylamides, N,N-dialkyl methacrylamides, Naryl methacrylamides,methacrylate esters, acrylate esters and functional equivalents thereof.

The polymer can be water soluble. This moiety is not limited by chemicalcontent. However, it simplifies analysis if the skeleton has arelatively reproducible size (for example, length, number of tag atoms,reproducible dendrimer character, etc.). The requirements for stability,solubility, and non-toxicity are also taken into consideration. Thus,the preparation and characterization of a functional water solublepolymer by a synthetic strategy that places many functional groups alongthe backbone plus a different reactive group (the linking group), thatcan be used to attach the polymer to a molecule (for example, an SBP),through a linker and optionally a spacer. The size of the polymer iscontrollable by controlling the polymerisation reaction. Typically thesize of the polymer will be chosen so as the radiation of gyration ofthe polymer is as small as possible, such as between 2 and 11nanometres. The length of an IgG antibody, an exemplary SBP, isapproximately 10 nanometres, and therefore an excessively large polymertag in relation to the size of the SBP may sterically interfere with SBPbinding to its target.

The metal-chelating group that is capable of binding at least one metalatom can comprise at least four acetic acid groups. For instance, themetal-chelating group can be a diethylenetriaminepentaacetate (DTPA)group or a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid(DOTA) group. Alternative groups include Ethylenediaminetetraacetic acid(EDTA) and ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraaceticacid (EGTA)

The metal-chelating group can be attached to the polymer through anester or through an amide. Examples of suitable metal-chelating polymersinclude the X8 and DM3 polymers available from Fluidigm Canada, Inc.

The polymer can be water soluble. Because of their hydrolytic stability,N-alkyl acrylamides, N-alkyl methacrylamides, and methacrylate esters orfunctional equivalents can be used. A degree of polymerization (DP) ofapproximately 1 to 1000 (1 to 2000 backbone atoms) encompasses most ofthe polymers of interest. Larger polymers are in the scope of theinvention with the same functionality and are possible as would beunderstood by practitioners skilled in the art. Typically the degree ofpolymerization will be between 1 and 10,000, such as 5-100, 10-250,250-5,000, 500-2,500, or 500-1,000. The polymers may be amenable tosynthesis by a route that leads to a relatively narrow polydispersity.The polymer may be synthesized by atom transfer radical polymerization(ATRP) or reversible addition-fragmentation (RAFT) polymerization, whichshould lead to values of Mw (weight average molecular weight)/Mn (numberaverage molecular weight) in the range of 1.1 to 1.2. An alternativestrategy involving anionic polymerization, where polymers with Mw/Mn ofapproximately 1.02 to 1.05 are obtainable. Both methods permit controlover end groups, through a choice of initiating or terminating agents.This allows synthesizing polymers to which the linker can be attached. Astrategy of preparing polymers containing functional pendant groups inthe repeat unit to which the liganded transition metal unit (for examplea Ln unit) can be attached in a later step can be adopted. Thisembodiment has several advantages. It avoids complications that mightarise from carrying out polymerizations of ligand containing monomers.

To minimize charge repulsion between pendant groups, the target ligandsfor (M³+) should confer a net charge of −1 on the chelate.

Polymers that be used in the invention include:

-   -   random copolymer poly(DMA-co-NAS): The synthesis of a 75/25 mole        ratio random copolymer of N-acryloxysuccinimide (NAS) with        N,N-dimethyl acrylamide (DMA) by RAFT with high conversion,        excellent molar mass control in the range of 5000 to 130,000,        and with Mw/Mn≈1.1 is reported in Relógio et al. (2004)        (Polymer, 45, 8639-49). The active NHS ester is reacted with a        metal-chelating group bearing a reactive amino group to yield        the metal-chelating copolymer synthesised by RAFT        polymerization.    -   poly(NMAS): NMAS can be polymerised by ATRP, obtaining polymers        with a mean molar mass ranging from 12 to 40 KDa with Mw/Mn of        approximately 1.1 (see e.g. Godwin et al., 2001; Angew.        Chem.Int.Ed, 40: 594-97).    -   poly(MAA): polymethacrylic acid (PMAA) can be prepared by        anionic polymerization of its t-butyl or trimethylsilyl (TMS)        ester.    -   poly(DMAEMA): poly(dimethylaminoethyl methacrylate) (PDMAEMA)        can be prepared by ATRP (see Wang et al, 2004, J.Am.Chem.Soc,        126, 7784-85). This is a well-known polymer that is conveniently        prepared with mean Mn values ranging from 2 to 35 KDa with Mw/Mn        of approximately 1.2 This polymer can also be synthesized by        anionic polymerization with a narrower size distribution.    -   polyacrylamide, or polymethacrylamide.

The metal-chelating groups can be attached to the polymer by methodsknown to those skilled in the art, for example, the pendant group may beattached through an ester or through an amide. For instance, to amethylacrylate based polymer, the metal-chelating group can be attachedto the polymer backbone first by reaction of the polymer withethylenediamine in methanol, followed by subsequent reaction of DTPAanhydride under alkaline conditions in a carbonate buffer.

A second means is to generate nanoparticles which can act as mass tags.A first pathway to generating such mass tags is the use of nanoscaleparticles of the metal which have been coated in a polymer. Here, themetal is sequestered and shielded from the environment by the polymer,and does not react when the polymer shell can be made to react e.g. byfunctional groups incorporated into the polymer shell. The functionalgroups can be reacted with linker components (optionally incorporating aspacer) to attach click chemistry reagents, so allowing this type ofmass tag to plug in to the synthetics strategies discussed above in asimple, modular fashion.

Grafting-to and grafting-from are the two principle mechanism forgenerating polymer brushes around a nanoparticle. In grafting to, thepolymers are synthesised separately, and so synthesis is not constrainedby the need to keep the nanoparticle colloidally stable. Here reversibleaddition-fragmentation chain transfer (RAFT) synthesis has excelled dueto a large variety of monomers and easy functionalization. The chaintransfer agent (CTA) can be readily used as functional group itself, afunctionalized CTA can be used or the polymer chains can bepost-functionalized. A chemical reaction or physisorption is used toattach the polymers to the nanoparticle. One drawback of grafting-to isthe usually lower grafting density, due to the steric repulsion of thecoiled polymer chains during attachment to the particle surface. Allgrafting-to methods suffer from the drawback that a rigorous workup isnecessary to remove the excess of free ligand from the functionalizednanocomposite particle. This is typically achieved by selectiveprecipitation and centrifugation. In the grafting-from approachmolecules, like initiators for atomic transfer radical polymerization(ATRP) or CTAs for (RAFT) polymerizations, are immobilized on theparticle surface. The drawbacks of this method are the development ofnew initiator coupling reactions. Moreover, contrary to grafting-to, theparticles have to be colloidally stable under the polymerizationconditions.

An additional means of generating a mass tag is via the use of dopedbeads. Chelated lanthanide (or other metal) ions can be employed inminiemulsion polymerization to create polymer particles with thechelated lanthanide ions embedded in the polymer. The chelating groupsare chosen, as is known to those skilled in the art, in such a way thatthe metal chelate will have negligible solubility in water butreasonable solubility in the monomer for miniemulsion polymerization.Typical monomers that one can employ are styrene, methylstyrene, variousacrylates and methacrylates, among others as is known to those skilledin the art. For mechanical robustness, the metal-tagged particles have aglass transition temperature (Tg) above room temperature. In someinstances, core-shell particles are used, in which the metal-containingparticles prepared by miniemulsion polymerization are used as seedparticles for a seeded emulsion polymerization to control the nature ofthe surface functionality. Surface functionality can be introducedthrough the choice of appropriate monomers for this second-stagepolymerization. Additionally, acrylate (and possible methacrylate)polymers are advantageous over polystyrene particles because the estergroups can bind to or stabilize the unsatisfied ligand sites on thelanthanide complexes. An exemplary method for making such doped beadsis: (a) combining at least one labelling atom-containing complex in asolvent mixture comprising at least one organic monomer (such as styreneand/or methyl methacrylate in one embodiment) in which the at least onelabelling atom-containing complex is soluble and at least one differentsolvent in which said organic monomer and said at least one labellingatom-containing complex are less soluble, (b) emulsifying the mixture ofstep (a) for a period of time sufficient to provide a uniform emulsion;(c) initiating polymerization and continuing reaction until asubstantial portion of monomer is converted to polymer; and (d)incubating the product of step (c) for a period of time sufficient toobtain a latex suspension of polymeric particles with the at least onelabelling atom-containing complex incorporated in or on the particlestherein, wherein said at least one labelling atom-containing complex isselected such that upon interrogation of the polymeric mass tag, adistinct mass signal is obtained from said at least one labelling atom.By the use of two or more complexes comprising different labellingatoms, doped beads can be made comprising two or more differentlabelling atoms. Furthermore, controlling the ration of the complexescomprising different labelling atoms, allows the production of dopedbeads with different ratios of the labelling atoms. By use of multiplelabelling atoms, and in different radios, the number of distinctivelyidentifiable mass tags is increased. In core-shell beads, this may beachieved by incorporating a first labelling atom-containing complex intothe core, and a second labelling atom-containing complex into the shell.

A yet further means is the generation of a polymer that include thelabelling atom in the backbone of the polymer rather than as aco-ordinated metal ligand. For instance, Carerra and Seferos(Macromolecules 2015, 48, 297-308) disclose the inclusion of telluriuminto the backbone of a polymer. Other polymers incorporating atomscapable as functioning as labelling atoms tin-, antimony- andbismuth-incorporating polymers. Such molecules are discussed inter aliain Priegert et al., 2016 (Chem. Soc. Rev., 45, 922-953).

Thus the mass tag can comprise at least two components: the labellingatoms, and a polymer, which either chelates, contains or is doped withthe labelling atom. In addition, the mass tag comprises an attachmentgroup (when not-conjugated to the SBP), which forms part of the chemicallinkage between the mass tag and the SBP following reaction of the twocomponents, in a click chemistry reaction in line with the discussionabove.

A polydopamine coating can be used as a further way to attach SBPs toe.g. doped beads or nanoparticles. Given the range of functionalities inpolydopamine, SBPs can be conjugated to the mass tag formed from a PDAcoated bead or particle by reaction of e.g. amine or sulhydryl groups onthe SBP, such as an antibody. Alternatively, the functionalities on thePDA can be reacted with reagents such as bifunctional linkers whichintroduce further functionalities in turn for reaction with the SBP. Insome instances, the linkers can contain spacers, as discussed below.These spacers increase the distance between the mass tag and the SBP,minimising steric hindrance of the SBP. Thus the invention comprises amass-tagged SBP, comprising an SBP and a mass tag comprisingpolydopamine, wherein the polydopamine comprises at least part of thelink between the SBP and the mass tag. Nanoparticles and beads, inparticular polydopamine coated nanoparticles and beads, may be usefulfor signal enhancement to detect low abundance targets, as they can havethousands of metal atoms and may have multiple copies of the sameaffinity reagent. The affinity reagent could be a secondary antibody,which could further boost signal.

Labelling Atom

Labelling atoms that can be used with the disclosure include any speciesthat are detectable by MS or OES and that are substantially absent fromthe unlabelled tissue sample. Thus, for instance, ¹²C atoms would beunsuitable as labelling atoms because they are naturally abundant,whereas ¹¹C could in theory be used for MS because it is an artificialisotope which does not occur naturally. Often the labelling atom is ametal. In preferred embodiments, however, the labelling atoms aretransition metals, such as the rare earth metals (the 15 lanthanides,plus scandium and yttrium). These 17 elements (which can bedistinguished by OES and MS) provide many different isotopes which canbe easily distinguished (by MS). A wide variety of these elements areavailable in the form of enriched isotopes e.g. samarium has 6 stableisotopes, and neodymium has 7 stable isotopes, all of which areavailable in enriched form. The 15 lanthanide elements provide at least37 isotopes that have non-redundantly unique masses. Examples ofelements that are suitable for use as labelling atoms include Lanthanum(La), Cerium (Ce), Praseodymium (Pr), Neodymium (Nd), Promethium (Pm),Samarium (Sm), Europium (Eu), Gadolinium, (Gd), Terbium (Tb), Dysprosium(Dy), Holmium (Ho), Erbium (Er), Thulium (Tm), Ytterbium (Yb), Lutetium(Lu), Scandium (Sc), and Yttrium (Y). In addition to rare earth metals,other metal atoms are suitable for detection e.g. gold (Au), platinum(Pt), iridium (Ir), rhodium (Rh), bismuth (Bi), etc. The use ofradioactive isotopes is not preferred as they are less convenient tohandle and are unstable e.g. Pm is not a preferred labelling atom amongthe lanthanides.

In order to facilitate time-of-flight (TOF) analysis (as discussedherein) it is helpful to use labelling atoms with an atomic mass withinthe range 80-250 e.g. within the range 80-210, or within the range100-200. This range includes all of the lanthanides, but excludes Sc andY. The range of 100-200 permits a theoretical 101-plex analysis by usingdifferent labelling atoms, while taking advantage of the high spectralscan rate of TOF MS. As mentioned above, by choosing labelling atomswhose masses lie in a window above those seen in an unlabelled sample(e.g. within the range of 100-200), TOF detection can be used to providerapid imaging at biologically significant levels.

Various numbers of labelling atoms can be attached to a single SBPmember dependent upon the mass tag used (and so the number of labellingatoms per mass tag) and the number of mass tags that are attached toeach SBP). Greater sensitivity can be achieved when more labelling atomsare attached to any SBP member. For example, greater than 10, 20, 30,40, 50, 60, 70, 80, 90 or 100 labelling atoms can be attached to a SBPmember, such as up to 10,000, for instance as 5-100, 10-250, 250-5,000,500-2,500, or 500-1,000 labelling atoms. As noted above, monodispersepolymers containing multiple monomer units may be used, each containinga chelator such as diethylenetriaminepentaacetic acid (DTPA) or DOTA.DTPA, for example, binds 3+ lanthanide ions with a dissociation constantof around 10³¹ ⁶ M. These polymers can terminate in a thiol which can beused for attaching to a SBP via reaction of that with a maleimide toattach a click chemistry reactivity in line with those discussed above.Other functional groups can also be used for conjugation of thesepolymers e.g. amine-reactive groups such as N-hydroxy succinimideesters, or groups reactive against carboxyls or against an antibody'sglycosylation. Any number of polymers may bind to each SBP. Specificexamples of polymers that may be used include straight-chain (“X8”)polymers or third-generation dendritic (“DN3”) polymers, both availableas MaxPar™ reagents. Use of metal nanoparticles can also be used toincrease the number of atoms in a label, as also discussed above.

In some embodiments, all labelling atoms in a mass tag are of the sameatomic mass. Alternatively, a mass tag can comprise labelling atoms ofdiffering atomic mass. Accordingly, in some instances, a labelled samplemay be labelled with a series of mass-tagged SBPs each of whichcomprises just a single type of labelling atom (wherein each SBP bindsits cognate target and so each kind of mass tag is localised on thesample to a specific e.g. antigen). Alternatively, in some instance, alabelled sample may be labelled with a series of mass-tagged SBPs eachof which comprises a mixture of labelling atoms. In some instances, themass-tagged SBPs used to label the sample may comprise a mix of thosewith single labelling atom mass tags and mixes of labelling atoms intheir mass tags.

Spacer

As noted above, in some instances, the SBP is conjugated to a mass tagthrough a linker which comprises a spacer. There may be a spacer betweenthe SBP and the click chemistry reagent (e.g. between the SBP and thestrained cycloalkyne (or azide); strained cycloalkene (or tetrazine);etc.). There may be a spacer between the between the mass tag and theclick chemistry reagent (e.g. between the mass tag and the azide (orstrained cycloalkyne); tetrazine (or strained cycloalkene); etc.). Insome instances there may be a spacer both between the SNP and the clickchemistry reagent, and the click chemistry reagent and the mass tag.

The spacer might be a polyethylene glycol (PEG) spacer, apoly(N-vinylpyrolide) (PVP) spacer, a polyglycerol (PG) spacer,poly(N-(2-hydroxylpropyl)methacrylamide) spacer, or a polyoxazoline(POZ, such as polymethyloxazoline, polyethyloxazoline orpolypropyloxazoline) or a C5-C20 non-cyclic alkyl spacer. For example,the spacer may be a PEG spacer with 3 or more, 4 or more, 5 or more, 6or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 ormore, 15 or more of 20 or more EG (ethylene glycol) units. The PEGlinker may have from 3 to 12 EG units, from 4 to 10, or may have 4, 5,6, 7, 8, 9, or 10 EG units. The linker may include cystamine orderivatives thereof, may include one or more disulfide groups, or may beany other suitable linker known to one of skill in the art.

Spacers may be beneficial to minimize the steric effect of the mass tagon the SBP to which is conjugated. Hydrophilic spacers, such as PEGbased spacers, may also act to improve the solubility of the mass-taggedSBP and act to prevent aggregation.

SBPs

Mass cytometry, including imaging mass cytometry is based on theprinciple of specific binding between members of specific binding pairs.The mass tag is linked to a specific binding pair member, and thislocalises the mass tag to the target/analyte which is the other memberof the pair. Specific binding does not require binding to just onemolecular species to the exclusion of others, however. Rather it definesthat the binding is not-nonspecific, i.e. not a random interaction. Anexample of an SBP that binds to multiple targets would therefore be anantibody which recognises an epitope that is common between a number ofdifferent proteins. Here, binding would be specific, and mediated by theCDRs of the antibody, but multiple different proteins would be detectedby the antibody. The common epitopes may be naturally occurring, or thecommon epitope could be an artificial tag, such as a FLAG tag.Similarly, for nucleic acids, a nucleic acid of defined sequence may notbind exclusively to a fully complementary sequence, but varyingtolerances of mismatch can be introduced under the use of hybridisationconditions of a differing stringencies, as would be appreciated by oneof skill in the art. Nonetheless, this hybridisation is notnon-specific, because it is mediated by homology between the SBP nucleicacid and the target analyte. Similarly, ligands can bind specifically tomultiple receptors, a facile example being TNFa which binds to bothTNFR1 and TNFR2.

The SBP may comprise any of the following: a nucleic acid duplex; anantibody/antigen complex; a receptor/ligand pair; or an aptamer/targetpair. Thus a labelling atom can be attached to a nucleic acid probewhich is then contacted with a tissue sample so that the probe canhybridise to complementary nucleic acid(s) therein e.g. to form aDNA/DNA duplex, a DNA/RNA duplex, or a RNA/RNA duplex. Similarly, alabelling atom can be attached to an antibody which is then contactedwith a tissue sample so that it can bind to its antigen. A labellingatom can be attached to a ligand which is then contacted with a tissuesample so that it can bind to its receptor. A labelling atom can beattached to an aptamer ligand which is then contacted with a tissuesample so that it can bind to its target. Thus, labelled SBP members canbe used to detect a variety of targets in a sample, including DNAsequences, RNA sequences, proteins, sugars, lipids, or metabolites.

The mass-tagged SBP therefore can be a protein or peptide, or apolynucleotide or oligonucleotide.

Examples of protein SBPs include an antibody or antigen binding fragmentthereof, a monoclonal antibody, a polyclonal antibody, a bispecificantibody, a multispecific antibody, an antibody fusion protein, scFv,antibody mimetic, avidin, streptavidin, neutravidin, biotin, or acombination thereof, wherein optionally the antibody mimetic comprises ananobody, affibody, affilin, affimer, affitin, alphabody, anticalin,avimer, DARPin, Fynomer, kunitz domain peptide, monobody, or anycombination thereof, a receptor, such as a receptor-Fc fusion, a ligand,such as a ligand-Fc fusion, a lectin, for example an agglutinin such aswheat germ agglutinin.

The peptide may be a linear peptide, or a cyclical peptide, such as abicyclic peptide. One example of a peptide that can be used isPhalloidin.

A polynucleotide or oligonucleotide generally refers to a single- ordouble-stranded polymer of nucleotides containing deoxyribonucleotidesor ribonucleotides that are linked by 3′-5′ phosphodiester bonds, aswell as polynucleotide analogs. A nucleic acid molecule includes, but isnot limited to, DNA, RNA, and cDNA. A polynucleotide analog may possessa backbone other than a standard phosphodiester linkage found in naturalpolynucleotides and, optionally, a modified sugar moiety or moietiesother than ribose or deoxyribose. Polynucleotide analogs contain basescapable of hydrogen bonding by Watson-Crick base pairing to standardpolynucleotide bases, where the analog backbone presents the bases in amanner to permit such hydrogen bonding in a sequence-specific fashionbetween the oligonucleotide analog molecule and bases in a standardpolynucleotide. Examples of polynucleotide analogs include, but are notlimited to xeno nucleic acid (XNA), bridged nucleic acid (BNA), glycolnucleic acid (GNA), peptide nucleic acids (PNAs), yPNAs, morpholinopolynucleotides, locked nucleic acids (LNAs), threose nucleic acid(TNA), 2′-0-Methyl polynucleotides, 2′-0-alkyl ribosyl substitutedpolynucleotides, phosphorothioate polynucleotides, and boronophosphatepolynucleotides. A polynucleotide analog may possess purine orpyrimidine analogs, including for example, 7-deaza purine analogs,8-halopurine analogs, 5-halopyrimidine analogs, or universal baseanalogs that can pair with any base, including hypoxanthine,nitroazoles, isocarbostyril analogues, azole carboxamides, and aromatictriazole analogues, or base analogs with additional functionality, suchas a biotin moiety for affinity binding.

Antibody SBP Members

In a typical embodiment, the labelled SBP member is an antibody.Labelling of the antibody can be achieved through conjugation of one ormore labelling atom binding molecules to the antibody, by attachment ofa mass tag using e.g. NHS-amine chemistry, sulfhydryl-maleimidechemistry, or the click chemistry (such as strained alkyne and azide,strained alkyne and nitrone, strained alkene and tetrazine etc.).Antibodies which recognise cellular proteins that are useful for imagingare already widely available for IHC usage, and by using labelling atomsinstead of current labelling techniques (e.g. fluorescence) these knownantibodies can be readily adapted for use in methods disclosure herein,but with the benefit of increasing multiplexing capability. Antibodiescan recognise targets on the cell surface or targets within a cell.Antibodies can recognise a variety of targets e.g. they can specificallyrecognise individual proteins, or can recognise multiple relatedproteins which share common epitopes, or can recognise specificpost-translational modifications on proteins (e.g. to distinguishbetween tyrosine and phosphor-tyrosine on a protein of interest, todistinguish between lysine and acetyl-lysine, to detect ubiquitination,etc.). After binding to its target, labelling atom(s) conjugated to anantibody can be detected to reveal the location of that target in asample.

The labelled SBP member will usually interact directly with a target SBPmember in the sample. In some embodiments, however, it is possible forthe labelled SBP member to interact with a target SBP member indirectlye.g. a primary antibody may bind to the target SBP member, and alabelled secondary antibody can then bind to the primary antibody, inthe manner of a sandwich assay. Usually, however, the method relies ondirect interactions, as this can be achieved more easily and permitshigher multiplexing. In both cases, however, a sample is contacted witha SBP member which can bind to a target SBP member in the sample, and ata later stage label attached to the target SBP member is detected.

Nucleic Acid SBPs, and Labelling Methodology Modifications

RNA is another biological molecule which the methods and apparatusdisclosed herein are capable of detecting in a specific, sensitive andif desired quantitative manner. In the same manner as described abovefor the analysis of proteins, RNAs can be detected by the use of a SBPmember labelled with an elemental tag that specifically binds to the RNA(e.g. an poly nucleotide or oligonucleotide of complementary sequence asdiscussed above, including a locked nucleic acid (LNA) molecule ofcomplementary sequence, a peptide nucleic acid (PNA) molecule ofcomplementary sequence, a plasmid DNA of complementary sequence, anamplified DNA of complementary sequence, a fragment of RNA ofcomplementary sequence and a fragment of genomic DNA of complementarysequence). RNAs include not only the mature mRNA, but also the RNAprocessing intermediates and nascent pre-mRNA transcripts.

In certain embodiments, both RNA and protein are detected using methodsof the claimed invention.

To detect RNA, cells in biological samples as discussed herein may beprepared for analysis of RNA and protein content using the methods andapparatus described herein. In certain aspects, cells are fixed andpermeabilized prior to the hybridization step. Cells may be provided asfixed and/or pemeabilized. Cells may be fixed by a crosslinkingfixative, such as formaldehyde, glutaraldehyde. Alternatively or inaddition, cells may be fixed using a precipitating fixative, such asethanol, methanol or acetone. Cells may be permeabilized by a detergent,such as polyethylene glycol (e.g., Triton X-100), Polyoxyethylene (20)sorbitan monolaurate (Tween-20), Saponin (a group of amphipathicglycosides), or chemicals such as methanol or acetone. In certain cases,fixation and permeabilization may be performed with the same reagent orset of reagents. Fixation and permeabilization techniques are discussedby Jamur et al. in “Permeabilization of Cell Membranes” (Methods Mol.Biol., 2010).

Detection of target nucleic acids in the cell, or “in-situhybridization” (ISH), has previously been performed usingfluorophore-tagged oligonucleotide probes. As discussed herein,mass-tagged oligonucleotides, coupled with ionization and massspectrometry, can be used to detect target nucleic acids in the cell.Methods of in-situ hybridization are known in the art (see Zenobi et al.“Single-Cell Metabolomics: Analytical and Biological Perspectives,”Science vol. 342, no. 6163, 2013). Hybridization protocols are alsodescribed in U.S. Pat. No. 5,225,326 and US Pub. No. 2010/0092972 and2013/0164750, which are incorporated herein by reference.

Prior to hybridization, cells present in suspension or immobilized on asolid support may be fixed and permeabilized as discussed earlier.Permeabilization may allow a cell to retain target nucleic acids whilepermitting target hybridization nucleotides, amplificationoligonucleotides, and/or mass-tagged oligonucleotides to enter the cell.The cell may be washed after any hybridization step, for example, afterhybridization of target hybridization oligonucleotides to nucleic acidtargets, after hybridization of amplification oligonucleotides, and/orafter hybridization of mass-tagged oligonucleotides.

Cells can be in suspension for all or most of the steps of the method,for ease of handling. However, the methods are also applicable to cellsin solid tissue samples (e.g., tissue sections) and/or cells immobilizedon a solid support (e.g., a slide or other surface). Thus, sometimes,cells can be in suspension in the sample and during the hybridizationsteps. Other times, the cells are immobilized on a solid support duringhybridization.

Target nucleic acids include any nucleic acid of interest and ofsufficient abundance in the cell to be detected by the subject methods.Target nucleic acids may be RNAs, of which a plurality of copies existwithin the cell. For example, 10 or more, 20 or more, 50 or more, 100 ormore, 200 or more, 500 or more, or 1000 or more copies of the target RNAmay be present in the cell. A target RNA may be a messenger NA (mRNA),ribosomal RNA (rRNA), transfer RNA (tRNA), small nuclear RNA (snRNA),small interfering RNA (siRNA), long noncoding RNA (IncRNA), or any othertype of RNA known in the art. The target RNA may be 20 nucleotides orlonger, 30 nucleotides or longer, 40 nucleotides or longer, 50nucleotides or longer, 100 nucleotides or longer, 200 nucleotides orlonger, 500 nucleotides or longer, 1000 nucleotides or longer, between20 and 1000 nucleotides, between 20 and 500 nucleotides in length,between 40 and 200 nucleotides in length, and so forth.

In certain embodiments, a mass-tagged oligonucleotide may be hybridizeddirectly to the target nucleic acid sequence. However, hybridization ofadditional oligonucleotides may allow for improved specificity and/orsignal amplification.

In certain embodiments, two or more target hybridizationoligonucleotides may be hybridized to proximal regions on the targetnucleic acid, and may together provide a site for hybridization of anadditional oligonucleotides in the hybridization scheme.

In certain embodiments, the mass-tagged oligonucleotide may behybridized directly to the two or more target hybridizationoligonucleotides. In other embodiments, one or more amplificationoligonucleotides may be added, simultaneously or in succession, so as tohybridize the two or more target hybridization oligonucleotides andprovide multiple hybridization sites to which the mass-taggedoligonucleotide can bind. The one or more amplificationoligonucleotides, with or without the mass-tagged oligonucleotide, maybe provided as a multimer capable of hybridizing to the two or moretarget hybridization oligonucleotides.

While the use of two or more target hybridization oligonucleotidesimproves specificity, the use of amplification oligonucleotidesincreases signal. Two target hybridization oligonucleotides arehybridized to a target RNA in the cell. Together, the two targethybridization oligonucleotides provide a hybridization site to which anamplification oligonucleotide can bind. Hybridization and/or subsequentwashing of the amplification oligonucleotide may be performed at atemperature that allows hybridization to two proximal targethybridization oligonucleotides, but is above the melting temperature ofthe hybridization of the amplification oligonucleotide to just onetarget hybridization oligonucleotide. The first amplificationoligonucleotide provides multiple hybridization sites, to which secondamplification oligonucleotides can be bound, forming a branched pattern.Mass-tagged oligonucleotides may bind to multiple hybridization sitesprovided by the second amplification nucleotides. Together, theseamplification oligonucleotides (with or without mass-taggedoligonucleotides) are referred to herein as a “multimer”. Thus the term“amplification oligonucleotide” includes oligonucleotides that providesmultiple copies of the same binding site to which furtheroligonucleotides can anneal. By increasing the number of binding sitesfor other oligonucleotides, the final number of labels that can be foundto a target is increased. Thus, multiple labelled oligonucleotides arehybridized, indirectly, to a single target RNA. This is enables thedetection of low copy number RNAs, by increasing the number ofdetectable atoms of the element used per RNA.

One particular method for performing this amplification comprises usingthe RNAscope® method from Advanced cell diagnostics, as discussed inmore detail below. A further alternative is the use of a method thatadapts the QuantiGene® FlowRNA method (Affymetrix eBioscience). Theassay is based on oligonucleotide pair probe design with branched DNA(bDNA) signal amplification. There are more than 4,000 probes in thecatalog or custom sets can be requested at no additional charge. In linewith the previous paragraph, the method works by hybridization of targethybridization oligonucleotides to the target, followed by the formationof a branched structure comprising first amplification oligonucleotides(termed preamplification oligonucleotides in the QuantiGene® method) toform a stem to which multiple second amplification oligonucleotides cananneal (termed simply amplification oligonucleotides in the QuantiGene®method). Multiple mass-tagged oligonucleotides can then bind.

Another means of amplification of the RNA signal relies on the rollingcircle means of amplification (RCA). There are various means why whichthis amplification system can be introduced into the amplificationprocess. In a first instance, a first nucleic acid is used as thehybridisation nucleic acid wherein the first nucleic acid is circular.The first nucleic acid can be single stranded or may be double-stranded.It comprises as sequence complementary to the target RNA. Followinghybridisation of the first nucleic acid to the target RNA, a primercomplementary to the first nucleic acid is hybridised to the firstnucleic acid, and used for primer extension using a polymerase andnucleic acids, typically exogenously added to the sample. In someinstances, however, when the first nucleic acid is added to sample, itmay already have the primer for extension hybridised to it. As a resultof the first nucleic acid being circular, once the primer extension hascompleted a full round of replication, the polymerase can displace theprimer and extension continues (i.e. without 5′→3′ exonuclase activity),producing linked further and further chained copies of the complement ofthe first nucleic acid, thereby amplifying that nucleic acid sequence.Oligonucleotides comprising an elemental tag (RNA or DNA, or LNA or PNAand the like) as discussed above) may therefore be hybridised to thechained copies of the complement of the first nucleic acid. The degreeof amplification of the RNA signal can therefore be controlled by thelength of time allotted for the step of amplification of the circularnucleic acid.

In another application of RCA, rather than the first, e.g.,oligonucleotide that hybridises to the target RNA being circular, it maybe linear, and comprise a first portion with a sequence complementary toits target and a second portion which is user-chosen. A circular RCAtemplate with sequence homologous to this second portion may then behybridised to this the first oligonucleotide, and RCA amplificationcarried out as above. The use of a first, e.g., oligonucleotide having atarget specific portion and user-chosen portion is that the user-chosenportion can be selected so as to be common between a variety ofdifferent probes. This is reagent-efficient because the same subsequentamplification reagents can be used in a series of reactions detectingdifferent targets. However, as understood by the skilled person, whenemploying this strategy, for individual detection of specific RNAs in amultiplexed reaction, each first nucleic acid hybridising to the targetRNA will need to have a unique second sequence and in turn each circularnucleic acid should contain unique sequence that can be hybridised bythe labelled oligonucleotide. In this manner, signal from each targetRNA can be specifically amplified and detected.

Other configurations to bring about RCA analysis will be known to theskilled person. In some instances, to prevent the first, e.g.,oligonucleotide dissociating from the target during the followingamplification and hybridisation steps, the first, e.g., oligonucleotidemay be fixed following hybridisation (such as by formaldehyde).

Further, hybridisation chain reaction (HCR) may be used to amplify theRNA signal (see, e.g., Choi et al., 2010, Nat. Biotech, 28:1208-1210).Choi explains that an HCR amplifier consists of two nucleic acid hairpinspecies that do not polymerise in the absence of an initiator. Each HCRhairpin consists of an input domain with an exposed single-strandedtoehold and an output domain with a single-stranded toehold hidden inthe folded hairpin. Hybridization of the initiator to the input domainof one of the two hairpins opens the hairpin to expose its outputdomain. Hybridization of this (previously hidden) output domain to theinput domain of the second hairpin opens that hairpin to expose anoutput domain identical in sequence to the initiator. Regeneration ofthe initiator sequence provides the basis for a chain reaction ofalternating first and second hairpin polymerization steps leading toformation of a nicked double-stranded ‘polymer’. Either or both of thefirst and second hairpins can be labelled with an elemental tag in theapplication of the methods and apparatus disclosed herein. As theamplification procedure relies on output domains of specific sequence,various discrete amplification reactions using separate sets of hairpinscan be performed independently in the same process. Thus thisamplification also permits amplification in multiplex analyses ofnumerous RNA species. As Choi notes, HCR is an isothermal triggeredself-assembly process. Hence, hairpins should penetrate the samplebefore undergoing triggered self-assembly in situ, suggesting thepotential for deep sample penetration and high signal-to-backgroundratios

Hybridization may include contacting cells with one or moreoligonucleotides, such as target hybridization oligonucleotides,amplification oligonucleotides, and/or mass-tagged oligonucleotides, andproviding conditions under which hybridization can occur. Hybridizationmay be performed in a buffered solution, such as saline sodium-citrate(SCC) buffer, phosphate-buffered saline (PBS), saline-sodiumphosphate-EDTA (SSPE) buffer, TNT buffer (having Tris-HCl, sodiumchloride and Tween 20), or any other suitable buffer. Hybridization maybe performed at a temperature around or below the melting temperature ofthe hybridization of the one or more oligonucleotides.

Specificity may be improved by performing one or more washes followinghybridization, so as to remove unbound oligonucleotide. Increasedstringency of the wash may improve specificity, but decrease overallsignal. The stringency of a wash may be increased by increasing ordecreasing the concentration of the wash buffer, increasing temperature,and/or increasing the duration of the wash. RNAse inhibitor may be usedin any or all hybridization incubations and subsequent washes.

A first set of hybridization probes, including one or more targethybridizing oligonucleotides, amplification oligonucleotides and/ormass-tagged oligonucleotides, may be used to label a first targetnucleic acid. Additional sets of hybridization probes may be used tolabel additional target nucleic acids. Each set of hybridization probesmay be specific for a different target nucleic acid. The additional setsof hybridization probes may be designed, hybridized and washed so as toreduce or prevent hybridization between oligonucleotides of differentsets. In addition, the mass-tagged oligonucleotide of each set mayprovide a unique signal. As such, multiple sets of oligonucleotides maybe used to detect 2, 3, 5, 10, 15, 20 or more distinct nucleic acidtargets.

Sometimes, the different nucleic acids detected are splice variants of asingle gene. The mass-tagged oligonucleotide can be designed tohybridize (directly or indirectly through other oligonucleotides asexplained below) within the sequence of the exon, to detect alltranscripts containing that exon, or may be designed to bridge thesplice junctions to detect specific variants (for example, if a gene hadthree exons, and two splice variants—exons 1-2-3 and exons 1-3—then thetwo could be distinguished: variant 1-2-3 could be detected specificallyby hybridizing to exon 2, and variant 1-3 could be detected specificallyby hybridizing across the exon 1-3 junction.

Histochemical Stains

The histochemical stain reagents having one or more intrinsic metalatoms may be combined with other reagents and methods of use asdescribed herein. For example, histochemical stains may be colocalized(e.g., at cellular or subcellular resolution) with metal containingdrugs, metal-labelled antibodies, and/or accumulated heavy metals. Incertain aspects, one or more histochemical stains may be used at lowerconcentrations (e.g., less than half, a quarter, a tenth, etc.) fromwhat is used for other methods of imaging (e.g., fluorescencemicroscopy, light microscopy, or electron microscopy).

To visualize and identify structures, a broad spectrum of histologicalstains and indicators are available and well characterized. Themetal-containing stains have a potential to influence the acceptance ofthe imaging mass cytometry by pathologists. Certain metal containingstains are well known to reveal cellular components, and are suitablefor use in the subject invention. Additionally, well defined stains canbe used in digital image analysis providing contrast for featurerecognition algorithms. These features are strategically important forthe development of imaging mass cytometry.

Often, morphological structure of a tissue section can be contrastedusing affinity products such as antibodies. They are expensive andrequire additional labelling procedure using metal-containin tags, ascompared to using histochemical stains. This approach was used inpioneering works on imaging mass cytometry using antibodies labelledwith available lanthanide isotopes thus depleting mass (e.g. metal) tagsfor functional antibodies to answer a biological question.

The subject invention expands the catalog of available isotopesincluding such elements as Ag, Au, Ru, W, Mo, Hf, Zr (includingcompounds such as Ruthenium Red used to identify mucinous stroma,Trichrome stain for identification of collagen fibers, osmium tetroxideas cell counterstain). Silver staining is used in karyotyping. Silvernitrate stains the nucleolar organization region (NOR)-associatedprotein, producing a dark region wherein the silver is deposited anddenoting the activity of rRNA genes within the NOR. Adaptation to IMCmay require that the protocols (e.g., oxidation with potassiumpermanganate and a silver concentration of 1% during) be modified foruse lower concentrations of silver solution, e.g., less than 0.5%,0.01%, or 0.05% silver solution.

Autometallographic amplification techniques have evolved into animportant tool in histochemistry. A number of endogenous and toxic heavymetals form sulfide or selenide nanocrystals that can beautocatalytically amplified by reaction with Ag ions. The larger Agnanocluster can then be readily visualized by IMC. At present, robustprotocols for the silver amplified detection of Zn—S/Se nanocrystalshave been established as well as detection of selenium through formationof silver-selenium nanocrystals. In addition, commercially availablequantum dots (detection of Cd) are also autocatalytically active and maybe used as histochemical labels.

Aspects of the subject invention may include histochemical stains andtheir use in imaging by elemental mass spectrometry. Any histochemicalstain resolvable by elemental mass spectrometry may be used in thesubject invention. In certain aspects, the histochemical stain includesone or more atoms of mass greater than a cut-off of the elemental massspectrometer used to image the sample, such as greater than 60 amu, 80amu, 100 amu, or 120 amu. For example, the histochemical stain mayinclude a metal tag (e.g., metal atom) as described herein. The metalatom may be chelated to the histochemical stain, or covalently boundwithin the chemical structure of the histochemical stain. In certainaspects, the histochemical stain may be an organic molecule.Histochemical stains may be polar, hydrophobic (e.g., lipophilic), ionicor may comprise groups with different properties. In certain aspects, ahistochemical stain may comprise more than one chemical.

Histochemical stains include small molecules of less than 2000, 1500,1000, 800, 600, 400, or 200 amu. Histochemical stains may bind to thesample through covalent or non-covalent (e.g., ionic or hydrophobic)interactions. Histochemical stains may provide contrast to resolve themorphology of the biological sample, for example, to help identifyindividual cells, intracellular structures, and/or extracellularstructures. Intracellular structures that may be resolved byhistochemical stains include cell membrane, cytoplasm, nucleus, Golgibody, ER, mitochondria, and other cellular organelles. Histochemicalstains may have an affinity for a type of biological molecule, such asnucleic acids, proteins, lipids, phospholipids or carbohydrates. Incertain aspects, a histochemical stain may bind a molecule other thanDNA. Suitable histochemical stains also include stains that bindextracellular structures (e.g., structures of the extracellular matrix),including stroma (e.g., mucosal stroma), basement membrane, interstitialstroma, proteins such as collage or elastin, proteoglycans,non-proteoglycan polysaccharides, extracellular vesicles, fibronectin,laminin, and so forth.

In certain aspects, histochemical stains and/or metabolic probes mayindicate a state of a cell or tissue. For example, histochemical stainsmay include vital stains such as cisplatin, eosin, and propidium iodide.Other histochemical stains may stain for hypoxia, e.g., may only bind ordeposit under hypoxic conditions. Probes such as lododeoxyuridine (IdU)or a derivative thereof, may stain for cell proliferation. In certainaspects, the histochemical stain may not indicate the state of the cellor tissue. Probes that detect cell state (e.g., viability, hypoxiaand/or cell proliferation) but are administered in-vivo (e.g., to aliving animal or cell culture) be used in any of the subject methods butdo not qualify as histochemical stains.

Histochemical stains may have an affinity for a type of biologicalmolecule, such as nucleic acids (e.g., DNA and/or RNA), proteins,lipids, phospholipids, carbohydrates (e.g., sugars such asmono-saccharides or di-saccharides or polyols; oligosaccharides; and/orpolysaccharides such as starch or glycogen), glycoproteins, and/orglycolipids. In certain aspects the histochemical stain may be acounterstain.

The following are examples of specific histochemical stains and theiruse in the subject methods:

Ruthenium Red stain as a metal-containing stain for mucinous stromadetection may be used as follows: Immunostained tissue (e.g.,de-paraffinized FFPE or cryosection) may be treated with 0.0001-0.5%,0.001-0.05%, less than 0.1%, less than 0.05%, or around 0.0025%Ruthenium Red (e.g., for at least 5 minutes, at least 10 minutes, atleast 30 minutes, or around 30 min at 4-42° C., or around roomtemperature). The biological sample may be rinsed, for example withwater or a buffered solution. Tissue may then be dried before imaging byelemental mass spectrometry.

Phosphotungstic Acid (e.g., as a Trichrome stain) may be used as ametal-containing stain for collagen fibers. Tissue sections on slides(de-paraffinized FFPE or cryosection) may be fixed in Bouin's fluid(e.g., for at least 5 minutes, at least 10 minutes, at least 30 minutes,or around 30 minutes at 4-42° C. or around room temperature). Thesections may then be treated with 0.0001%-0.01%, 0.0005%-0.005%, oraround 0.001% Phosphotangstic Acid for (e.g., for at least 5 minutes, atleast 10 minutes, at least 30 minutes, or around 15 minutes at 4-42° C.or around room temperature). Sample may then be rinsed with water and/orbuffered solution, and optionally dried, prior to imaging by elementalmass spectrometry. Triichrome stain may be used ata dilution (e.g., 5fold, 10 fold, 20 fold, 50 fold or great dilution) compared toconcentrations used for imaging by light (e.g., fluorescence)microscopy.

In some embodiments, the histochemical stain is an organic molecule. Insome embodiments, the second metal is covalently bound. In someembodiments, the second metal is chelated. In some embodiments, thehistochemical stain specifically binds cell membrane. In someembodiments, the histochemical stain is osmium tetroxide. In someembodiments, the histochemical stain is lipophilic. In some embodiments,the histochemical stain specifically binds an extracellular structure.In some embodiments, the histochemical stain specifically bindsextracellular collagen. In some embodiments, the histochemical stain isa trichrome stain comprising phosphotungstic/phosphomolybdic acid. Insome embodiments, trichrome stain is used after contacting the samplewith the antibody, such as at a lower concentration than would be usedfor optical imaging, for instance wherein the concentration is a 50 folddilution of trichrome stain or greater.

Metal-Containing Drugs

Metals in medicine is a new and exciting field in pharmacology. Littleis known about the cellular structures that are involved in transientlystoring metal ions prior to their incorporation into metalloproteins,nucleic acid metal complexes or metal-containing drugs or the fate ofmetal ions upon protein or drug degradation. An important first steptowards unravelling the regulatory mechanisms involved in trace metaltransport, storage, and distribution represents the identification andquantitation of the metals, ideally in context of their nativephysiological environment in tissues, cells, or even at the level ofindividual organelles and subcellular compartments. Histological studiesare typically carried out on thin sections of tissue or with culturedcells.

A number of metal-containing drugs are being used for treatment ofvarious diseases, however not enough is known about their mechanism ofaction or biodistribution: cisplatin, ruthenium imidazole,metallocene-based anti-cancer agents with Mo, tungstenocenes with W,B-diketonate complexes with Hf or Zr, auranofin with Au,polyoxomolybdate drugs. Many metal complexes are used as MRI contrastagents (Gd(III) chelates). Characterization of the uptake andbiodistribution of metal-based anti-cancer drugs is of criticalimportance for understanding and minimizing the underlying toxicity.

The atomic masses of certain metals present in drugs fall into the rangeof mass cytometry. Specifically, cisplatin and others with Pt complexes(iproplatin, lobplatin) are extensively used as a chemotherapeutic drugfor treating a wide range of cancers. The nephrotoxicity andmyelotoxicity of platinum-based anti-cancer drugs is well known. Withthe methods and reagents described herein, their subcellularlocalization within tissue sections, and colocalization with mass-(e.g.metal-) tagged antibodies and/or histochemical stains can now beexamined. Chemotherepeutic drugs may be toxic to certain cells, such asproliferating cells, through direct DNA damage, inhibition of DNA damagerepair pathways, radioactivity, and so forth. In certain aspects,chemotherapeutic drugs may be targeted to tumor through an antibodyintermediate.

In certain aspects, the metal containing drug is a chemotherapeuticdrug. Subject methods may include administering the metal containingdrug to a living animal, such as an animal research model or humanpatient as previously described, prior to obtaining the biologicalsample. The biological sample may be, for example, a biopsy of canceroustissue or primary cells. Alternatively, the metal containing drug may beadded directly to the biological sample, which may be an immortalizedcell line or primary cells. When the animal is a human patient, thesubject methods may include adjusting a treatment regimen that includesthe metal containing drug, based on detecting the distribution of themetal containing drug.

The method step of detecting the metal containing drug may includesubcellular imaging of the metal containing drug by elemental massspectrometry, and may include detecting the retention of the metalcontaining drug in an intracellular structure (such as membrane,cytoplasm, nucleus, Golgi body, ER, mitochondria, and other cellularorganelles) and/or extracellular structure (such as including stroma,mucosal stroma, basement membrane, interstitial stroma, proteins such ascollage or elastin, proteoglycans, non-proteoglycan polysaccharides,extracellular vesicles, fibronectin, laminin, and so forth).

A histochemical stain and/or mass-(e.g. metal-) tagged SBP that resolves(e.g., binds to) one or more of the above structures may be colocalizedwith the metal containing drug to detected retention of the drug atspecific intracellular or extracellular structures. For example, achemotherapeutic drug such as cisplatin may be colocalized with astructure such as collagen. Alternatively or in addition, thelocalization of the drug may be related to presence of a marker of cellviability, cell proliferation, hypoxia, DNA damage response, or immuneresponse.

In some embodiments, the metal containing drug comprises anon-endogenous metal, such as wherein the non-endogenous metal isplatinum, palladium, cerium, cadmium, silver or gold. In certainaspects, the metal containing drug is one of cisplatin, rutheniumimidazole, metallocene-based anti-cancer agents with Mo, tungstenoceneswith W, B-diketonate complexes with Hf or Zr, auranofin with Au,polyoxomolybdate drugs, N-myristoyltransferase-1 inhibitor(Tris(dibenzylideneacetone) dipalladium) with Pd, or a derivativethereof. For example the drug may comprise Pt, and may be, for example,cisplatin, carboplatin, oxaliplatin, iproplatin, lobaplatin or aderivative thereof. The metal containing drug may include anon-endogenous metal, such as platinum (Pt), ruthenium (Ru), molybdenum(Mo), tungsten (W), hafnium (Hf), zirconium (Zr), gold (Au), gadolinium(Gd), palladium (Pd) or an isotope thereof. Gold compounds (Auranofin,for example) and gold nanoparticle bioconjugates for photothermaltherapy against cancer can be identified in tissue sections.

Multiplexed Analysis

One feature of the disclosure is its ability to detect multiple (e.g. 10or more, and even up to 100 or more) different target SBP members in asample e.g. to detect multiple different proteins and/or multipledifferent nucleic acid sequences. To permit differential detection ofthese target SBP members their respective SBP members should carrydifferent labelling atoms such that their signals can be distinguished.For instance, where ten different proteins are being detected, tendifferent antibodies (each specific for a different target protein) canbe used, each of which carries a unique label, such that signals fromthe different antibodies can be distinguished. In some embodiments, itis desirable to use multiple different antibodies against a singletarget e.g. which recognise different epitopes on the same protein.Thus, a method may use more antibodies than targets due to redundancy ofthis type. In general, however, the disclosure will use a plurality ofdifferent labelling atoms to detect a plurality of different targets.

If more than one labelled antibody is used with the disclosure, it ispreferable that the antibodies should have similar affinities for theirrespective antigens, as this helps to ensure that the relationshipbetween the quantity of labelling atoms detected and the abundance ofthe target antigen in the tissue sample will be more consistent acrossdifferent SBPs (particularly at high scanning frequencies). Similarly,it is preferable if the labelling of the various antibodies has the sameefficiency, so that the antibodies each carry a comparable quantity ofthe labelling atom.

In some instances, the SBP may carry a fluorescent label as well as anelemental tag. Fluorescence of the sample may then be used to determineregions of the sample, e.g. a tissue section, comprising material ofinterest which can then be sampled for detection of labelling atoms.E.g. a fluorescent label may be conjugated to an antibody which binds toan antigen abundant on cancer cells, and any fluorescent cell may thenbe targeted to determine expression of other cellular proteins that areabout by SBPs conjugated to labelling atoms.

If a target SBP member is located intracellularly, it will typically benecessary to permeabilize cell membranes before or during contacting ofthe sample with the labels. For example, when the target is a DNAsequence but the labelled SBP member cannot penetrate the membranes oflive cells, the cells of the tissue sample can be fixed andpermeabilised. The labelled SBP member can then enter the cell and forma SBP with the target SBP member. In this respect, known protocols foruse with IHC and FISH can be utilised.

A method may be used to detect at least one intracellular target and atleast one cell surface target. In some embodiments, however, thedisclosure can be used to detect a plurality of cell surface targetswhile ignoring intracellular targets. Overall, the choice of targetswill be determined by the information which is desired from the method,as the disclosure will provide an image of the locations of the chosentargets in the sample.

As described further herein, specific binding partners (i.e., affinityreagents) comprising labelling atoms may be used to stain (contact) abiological sample. Suitable specific binging partners include antibodies(including antibody fragments). Labelling atoms may be distinguishableby mass spectrometry (i.e., may have different masses). Labelling atomsmay be referred to herein as metal tags when they include one or moremetal atoms. Metal tags may include a polymer with a carbon backbone anda plurality of pendant groups that each bind a metal atom.Alternatively, or in addition, metal tags may include a metalnanoparticle. Antibodies may be tagged with a metal tag by a covalent ornon-covalent interaction.

Antibody stains may be used to image proteins at cellular or subcellularresolution. Aspects of the invention include contacting the sample withone or more antibodies that specifically bind a protein expressed bycells of the biological sample, wherein the antibody is tagged with afirst metal tag. For example, the sample may be contacted with 5 ormore, 10 or more, 15 or more, 20 or more, 30 or more, 40 or more, 50 ormore antibodies, each with a distinguishable metal tag. The sample mayfurther be contacted with one or more histochemical stains before,during (e.g., for ease of workflow), or after (e.g., to avoid alteringantigen targets of antibodies) staining the sample with antibodies. Thesample may further comprise one or more metal containing drugs and/oraccumulated heavy metals as described herein.

Metal tagged antibodies for use in the subject inventions mayspecifically bind a metabolic probe that does not comprise a metal(e.g., EF5). Other metal tagged antibodies may specifically bind atarget (e.g., of epithelial tissue, stromal tissue, nucleus, etc.) oftraditional stains used in fluorescence and light microscopy. Suchantibodies include anti-cadherin, anti-collagen, anti-keratin, anti-EFS,anti-Histone H3 antibodies, and a number of other antibodies known inthe art.

Common histochemical stains that can be used herein include RutheniumRed and Phosphotungstic Acid (e.g., as a Trichrome stain).

In addition to specific staining of sample introduce a stain into thesample, sometimes, the sample may contain a metal atom as a result ofthe tissue or the organism from which it was taken being administered ametal containing drug, or having accumulated metals from environmentalexposure. Sometimes, tissues or animals may be tested in methods usingthis technique based on a pulse chase experimental strategy, to observeretention and clearance of a metal-containing material.

For instance, metals in medicine is a new and exciting field inpharmacology. Little is known about the cellular structures that areinvolved in transiently storing metal ions prior to their incorporationinto metalloproteins, nucleic acid metal complexes or metal-containingdrugs or the fate of metal ions upon protein or drug degradation. Animportant first step towards unravelling the regulatory mechanismsinvolved in trace metal transport, storage, and distribution representsthe identification and quantification of the metals, ideally in contextof their native physiological environment in tissues, cells, or even atthe level of individual organelles and subcellular compartments.Histological studies are typically carried out on thin sections oftissue or with cultured cells.

A number of metal-containing drugs are being used for treatment ofvarious diseases, however not enough is known about their mechanism ofaction or biodistribution: cisplatin, ruthenium imidazole,metallocene-based anti-cancer agents with Mo, tungstenocenes with W,B-diketonate complexes with Hf or Zr, auranofin with Au,polyoxomolybdate drugs. Many metal complexes are used as MRI contrastagents (Gd(III) chelates). Characterization of the uptake andbiodistribution of metal-based anti-cancer drugs is of criticalimportance for understanding and minimizing the underlying toxicity.

The atomic masses of certain metals present in drugs fall into the rangeof mass cytometry. Specifically, cisplatin and others with Pt complexes(iproplatin, lobplatin) are extensively used as a chemotherapeutic drugfor treating a wide range of cancers. The nephrotoxicity andmyelotoxicity of platinum-based anti-cancer drugs is well known. Withthe methods and reagents described herein, their subcellularlocalization within tissue sections, and colocalization with metaltagged antibodies and/or histochemical stains can now be examined.Chemotherepeutic drugs may be toxic to certain cells, such asproliferating cells, through direct DNA damage, inhibition of DNA damagerepair pathways, radioactivity, and so forth. In certain aspects,chemotherapeutic drugs may be targeted to tumor through an antibodyintermediate.

In certain aspects, the metal containing drug is a chemotherapeuticdrug. Subject methods may include administering the metal containingdrug to a living animal, such as an animal research model or humanpatient as previously described, prior to obtaining the biologicalsample. The biological sample may be, for example, a biopsy of canceroustissue or primary cells. Alternatively, the metal containing drug may beadded directly to the biological sample, which may be an immortalizedcell line or primary cells. When the animal is a human patient, thesubject methods may include adjusting a treatment regimen that includesthe metal containing drug, based on detecting the distribution of themetal containing drug.

The method step of detecting the metal containing drug may includesubcellular imaging of the metal containing drug by elemental massspectrometry, and may include detecting the retention of the metalcontaining drug in an intracellular structure (such as membrane,cytoplasm, nucleus, Golgi body, ER, mitochondria, and other cellularorganelles) and/or extracellular structure (such as including stroma,mucosal stroma, basement membrane, interstitial stroma, proteins such ascollage or elastin, proteoglycans, non-proteoglycan polysaccharides,extracellular vesicles, fibronectin, laminin, and so forth).

A histochemical stain and/or metal-tagged SBP that resolves (e.g., bindsto) one or more of the above structures may be colocalized with themetal containing drug to detected retention of the drug at specificintracellular or extracellular structures. For example, achemotherapeutic drug such as cisplatin may be colocalized with astructure such as collagen. Alternatively or in addition, thelocalization of the drug may be related to presence of a marker of cellviability, cell proliferation, hypoxia, DNA damage response, or immuneresponse.

In certain aspects, the metal containing drug is one of cisplatin,ruthenium imidazole, metallocene-based anti-cancer agents with Mo,tungstenocenes with W, B-diketonate complexes with Hf or Zr, auranofinwith Au, polyoxomolybdate drugs, N-myristoyltransferase-1 inhibitor(Tris(dibenzylideneacetone) dipalladium) with Pd, or a derivativethereof. For example the drug may comprise Pt, and may be, for example,cisplatin, carboplatin, oxaliplatin, iproplatin, lobaplatin or aderivative thereof. The metal containing drug may include anon-endogenous metal, such as platinum (Pt), ruthenium (Ru), molybdenum(Mo), tungstein (W), hafnium (Hf), zirconium (Zr), gold (Au), gadolinium(Gd), palladium (Pd) or an isotope thereof. Gold compounds (Auranofin,for example) and gold nanoparticle bioconjugates for photothermaltherapy against cancer can be identified in tissue sections.

Exposure to heavy metals can occur though ingestion of food or water,contact through skin, or aerosol intake. Heavy metals may accumulate insoft tissues of the body, such that prolonged exposure has serioushealth effects. In certain aspect, the heavy metal may be accumulated invivo, either through controlled exposure in an animal research model orthough environmental exposure in a human patient. The heavy metal may bea toxic heavy metal, such as Arsenic (As), Lead (Pb), Antimony (Sb),Bismuth (Bi), Cadmium (Cd), Osmium (Os), Thallium (TI), or Mercury (Hg).

Single Cell Analysis

Methods of the disclosure include laser ablation of multiple cells in asample, and thus plumes from multiple cells are analysed and theircontents are mapped to specific locations in the sample to provide animage. In most cases a user of the method will need to localise thesignals to specific cells within the sample, rather than to the sampleas a whole. To achieve this, the boundaries of cells (e.g. the plasmamembrane, or in some cases the cell wall) in the sample can bedemarcated.

Demarcation of cellular boundaries can be achieved in various ways. Forinstance, a sample can be studied using conventional techniques whichcan demarcate cellular boundaries, such as microscopy as discussedabove. When performing these methods, therefore, an analysis systemcomprising a camera as discussed above is particularly useful. An imageof this sample can then be prepared using a method of the disclosure,and this image can be superimposed on the earlier results, therebypermitting the detected signals to be localised to specific cells.Indeed, as discussed above, in some cases the laser ablation may bedirected only to a subset of cells in the sample as determined to be ofinterest by the use of microscopy based techniques.

To avoid the need to use multiple techniques, however, it is possible todemarcate cellular boundaries as part of the imaging method of thedisclosure. Such boundary demarcation strategies are familiar from IHCand immunocytochemistry, and these approaches can be adapted by usinglabels which can be detected. For instance, the method can involvelabelling of target molecule(s) which are known to be located atcellular boundaries, and signal from these labels can then be used forboundary demarcation. Suitable target molecules include abundant oruniversal markers of cell boundaries, such as members of adhesioncomplexes (e.g. β-catenin or E-cadherin). Some embodiments can labelmore than one membrane protein in order to enhance demarcation.

In addition to demarcating cell boundaries by including suitable labels,it is also possible to demarcate specific organelles in this way. Forinstance, antigens such as histones (e.g. H3) can be used to identifythe nucleus, and it is also possible to label mitochondrial-specificantigens, cytoskeleton-specific antigens, Golgi-specific antigens,ribosome-specific antigens, etc., thereby permitting cellularultrastructure to be analysed by methods of the disclosure.

Signals which demarcate the boundary of a cell (or an organelle) can beassessed by eye, or can be analysed by computer using image processing.Such techniques are known in the art for other imaging techniques e.g.Arce et al. (2013; Scientific Reports 3, article 2266) describes asegmentation scheme that uses spatial filtering to determine cellboundaries from fluorescence images, Ali et al. (2011; Mach VisApp/23:607-21) discloses an algorithm which determines boundaries frombrightfield microscopy images, Pound et al. (2012; The Plant Cell24:1353-61) discloses the CelISeT method to extract cell geometry fromconfocal microscope images, and Hodneland et al. (2013; Source Code forBiology and Medicine 8:16) discloses the CellSegm MATLAB toolbox forfluorescence microscope images. A method which is useful with thedisclosure uses watershed transformation and Gaussian blurring. Theseimage processing techniques can be used on their own, or they can beused and then checked by eye.

Once cellular boundaries have been demarcated it is possible to allocatesignal from specific target molecules to individual cells. It can alsobe possible to quantify the amount of a target analyte(s) in anindividual cell e.g. by calibrating the methods against quantitativestandards.

Reference Particles

As described herein, reference particles of known elemental or isotopiccomposition may be added to the sample (or the sample carrier) for useas a reference during detection of target elemental ions in the sample.In certain embodiments, reference particles comprise metal elements orisotopes, such as transition metals or lanthanides. For example,reference particles may comprise elements or isotopes of mass greaterthan 60 amu, greater than 80 amu, greater than 100 amu, or greater than120 amu.

Target elements, such as labelling atoms, can be normalized within asample run based on elemental ions detected from individual referenceparticles. For example, the subject methods may include switchingbetween detecting elemental ions from individual reference particles anddetecting only target elemental ions.

Pre-Analysis Sample Expansion Using Hydrogels

Conventional light microscopy is limited to approximately half thewavelength of the source of illumination, with a minimum possibleresolution of about 200 nm. Expansion microscopy is a method of samplepreparation (in particular for biological samples) that uses polymernetworks to physically expand the sample and so increase the resolutionof optical visualisation of a sample to around 20 nm (WO2015127183). Theexpansion procedures can be used to prepare samples for imaging massspectrometry and imaging mass cytometry. By this process, a 1 μmablation spot diameter would provide a resolution of 1 μm on anunexpanded sample, but with this 1 μm ablation spot represents ˜100 nmresolution following expansion. In certain aspects, the size of theablation spot (e.g., spot size) may be less than the size of the sampleimpinged by the radiation (e.g., laser, ion or electron beam radiation),for example when the energy required for ablation is greater than theenergy at the edge of the beam impinging the sample.

Expansion microscopy of biological samples generally comprises the stepsof: fixation, preparation for anchoring, gelation, mechanicalhomogenization, and expansion.

In the fixation stage, samples chemically fixed and washed. However,specific signalling functions or enzymatic functions such asprotein-protein interactions as a function of physiological state can beexamined using expansion microscopy without a fixation step.

Next, the samples are prepared so that they can be attached (“anchored”)to the hydrogel formed in the subsequent gelation step. Here, SBPs asdiscussed elsewhere herein (e.g. an antibody, nanobody, non-antibodyprotein, peptide, nucleic acid and/or small molecule that canspecifically bind to target molecules of interest in the sample) areincubated with the sample to bind to the targets if present in thesample. Optionally, samples can be labelled (sometimes termed‘anchored’) with a detectable compound useful for imaging. For opticalmicroscopy, the detectable compound could comprise, for example, beprovided by a fluorescently labelled antibody, nanobody, non-antibodyprotein, peptide, nucleic acid and/or small molecule that canspecifically bind to target molecules of interest in the sample(US2017276578). For mass cytometry, including imaging mass cytometry,the detectable label could be provided by, for example, an elemental taglabelled antibody, nanobody, non-antibody protein, peptide, nucleic acidand/or small molecule that can specifically bind to target molecules ofinterest in the sample. In some instances, the SBP binding to the targetdoes not contain a label but instead contains a feature that can bebound by a secondary SBP (e.g. a primary antibody that binds to thetarget and a secondary antibody that binds to the primary antibody, ascommon in immunohistochemical techniques). If only a primary SBP isused, this may itself be linked to a moiety that attaches or crosslinksthe sample to the hydrogel formed in the subsequent gelation step sothat the sample can be tethered to the hydrogel. Alternatively, if asecondary SBP is used, this may contain the moiety that attaches orcrosslinks the sample to the hydrogel. In some instances, a third SBP isused, which binds to the secondary SBP. One exemplary experimentalprotocol is set out in Chen et al., 2015 (Science 347: 543-548) uses aprimary antibody to bind to the target, a secondary antibody that bindsto the primary antibody wherein the secondary antibody is attached to anoligonucleotide sequence, and then as a tertiary SBP a oligonucleotidecomplementary to the sequence attached to the secondary antibody,wherein the tertiary SBP comprised a methacryloyl group that can beincorporated into an acrylamide hydrogel. In some instances, the SBPcomprising the moiety that is incorporated into the hydrogel alsoincludes a label. These labels can be fluorescent labels or elementaltags and so used in subsequent analysis by, for example, flow cytometry,optical scanning and fluorometry (US2017253918), or mass cytometry orimaging mass cytometry.

The gelation stage generates a matrix in the sample, by infusing ahydrogel comprising densely cross-linked, highly charged monomers intothe sample. For example, sodium acrylate along with the comonomeracrylamide and the crosslinker N-N′methylenebisacrylamide have beenintroduced into fixed and permeablised brain tissue (see Chen et al.,2015). When the polymer forms, it incorporates the moiety linked to thetargets in the anchoring step, so that the targets in the sample becomeattached to the gel matrix.

The sample is then treated with a homogenizing agent to homogenize themechanical characteristics of the sample so that the sample does notresist expansion (WO2015127183). For example, the sample can behomogenised by degradation with an enzyme (such as a protease), bychemical proteolysis, (e.g. by cyanogen bromide), by heating of thesample to 70-95 degrees Celsius, or by physical disruption such assonication (US2017276578).

The sample/hydrogel composite is then expanded by dialyzing thecomposite in a low-salt buffer or water to allow the sample to expand to4× or 5× its original size in 3-dimensions. As the hydrogel expands, sodoes the sample and in particular the labels attached to targets and thehydrogel expand, while maintaining their original three dimensionalarrangement of the labels. Since the samples expand are expanded inlow-salt solutions or water, the expanded samples are clear, allowingoptical imaging deep into the samples, and allow imaging withoutintroduction of significant levels of contaminating elements whenperforming mass cytometry (e.g. by use of distilled water or purified byother processes including capacitive deionization, reverse osmosis,carbon filtering, microfiltration, ultrafiltration, ultravioletoxidation, or electrodeionization).

The expanded sample can then be analysed by imaging techniques,providing pseudo-improved resolution. For example, fluorescencemicroscopy can be used with fluorescent labels, and imaging masscytometry can be used with elemental tags, optionally in combination.Due to the swelling of the hydrogel and the concomitant increase indistance between labels in the expanded sample vis-à-vis the nativesample, labels which were not capable of being resolved separatelypreviously (be that due to diffraction limit of visible light in opticalmicroscopy, or spot diameter in IMC).

Variants of expansion microscopy (ExM) exist, which can also be appliedusing the apparatus and methods disclosed herein. These variantsinclude: protein retention ExM (proExM), expansion fluorescent in situhybridisation (ExFISH), iterative ExM (iExM),Iterative expansionmicroscopy involves forming a second expandable polymer gel in a samplethat has already undergone a preliminary expansion using the abovetechniques. The first expanded gel is dissolved and the secondexpandable polymer gel is then expanded to bring the total expansion toup to ˜20×. For instance, Chang et al., 2017 (Nat Methods 14:593-599)base the technique on the method of Chen et al. 2015 discussed above,with the substitution that the first gel is made with a cleavable crosslinker (e.g., the commercially available crosslinkerN,N′-(1,2-dihydroxyethylene) bisacrylamide (DHEBA), whose diol bond canbe cleaved at high pH). Following anchoring and expansion of the firstgel, a labelled oligonucleotide (comprising a moiety for incorporationinto a second gel) and complementary to the oligonucleotide incorporatedinto the first gel was added to the expanded sample. A second gel wasformed incorporating the moiety of the labelled oligonucleotide, and thefirst gel was broken down by cleavage of the cleavable linker. Thesecond gel was then expanded in the same manner as the first, resultingin further spatial separation of the labels, but maintaining theirspatial arrangement with respect to the arrangement of the targets inthe original sample. In some instances, following expansion of the firstgel, an intermediate “re-embedding gel” is used, to hold the expandedfirst gel in place while the experimental steps are undertaken, e.g., tohybridise the labelled SBP to the first gel matrix, form the unexpandedsecond hydrogel, before the first hydrogel and the re-embedding gel arebroken down to permit the expansion of the second hydrogel. As beforethe labels used can be fluorescent or elemental tags and so used insubsequent analysis by, for example, flow cytometry, optical scanningand fluorometry, or mass cytometry or imaging mass cytometry, asappropriate.

Parameters and Applications

Methods and systems described herein may achieve a spot size diameter ator less than 500 nm, 400 nm, 300 nm, 200 nm, 100 nm, 50 nm, 30 nm, or 20nm in diameter. In addition, the depth of the spot may be shallow; thespot depth may be similar to the diameter of the spot size or may beless than the diameter of the spot size. For example, a fs laser mayablate at a shallow depth (for example, when directed at the sample fromthe same side as plume formation, or if focused slightly above thesample when directed through a transparent substrate). A shallow spotdepth may be at or less than 100 nm, 50 nm, 30 nm, 20 nm, or 10 nm. Assuch, some spots may only comprise one copy of a mass tag (e.g., oneinstance of a mass tag attached to a target, such as an RNA or protein).The mass tag may be attached to the target through an antibodyintermediate and/or a hybridized oligonucleotide probe. In some cases,the same location (e.g., X-Y coordinate) may be sampled multiple times(e.g., at different Z-depths), improving detection by acquiring more ofthe sample and/or allowing for 3D imaging.

Resolution could be further improved through expansion of the sample(e.g., as expansion of a matrix crosslinking the sample such as by gelexpansion).

Further, methods and systems described herein may provide improvedionization efficiency (such as by laser-SNMS, electron seeding prior toablation, laser ionization post ablation, and/or laser ionization postinitial neutralization). A postionization efficiency (percentage oflabeling atoms ionized that remain charged through detection) may be ator above 5%, 10%, 20%, 30% or 50%. Further, formation of ions at or nearthe sample surface allow such ions to be transported directly by ionoptics directly to a mass spectrometer, without losses incurred duringfluidic transport of an ablation plume to ICP.

Alternatively or in addition, sensitivity may be improved with mass tagsthat comprise a large number of labeling atoms. Such a mass tag maycomprise a metal chelating polymer, a metal nanoparticle (e.g., ametallocrystal or metal entrapped in a polymer matrix), or ahybridization scheme in which a plurality of metal taggedoligonucleotides hybridized to a RNA target or to an oligonucleotideconjugated to an affinity reagent (e.g., antibody) bound to a targetprotein. A high sensitivity mass tag may have at least 20, 50, 100, 200,500, 1000, 2000, 5000, or 10000 labeling atoms.

As such, the methods and systems described herein may allow fordetection of a single mass tag in a single spot (e.g., pixel). Whensingle mass tags can be detected, the mass tag may comprise a uniquecombination of labeling atoms that are specific for the target bound bythe mass tag (i.e., a target barcode). This may increase the number oftargets that can be distinguished beyond the number of differentlabeling atom masses. A target barcode may have enriched isotopes from aplurality of different elements. For example, mass tags that comprise 5out of 10 different labeling atoms allows for 10 choose 5 (i.e., 252)unique target barcodes. Mass tags that comprise 10 out of 20 differentlabeling atoms allows for 20 choose 10 (i.e., 184,756) unique targetbarcodes. As such, more than 50, 100, 200, 500, 1000, 2000, 5000, or10000 different targets may be target barcoded. If target barcodescomprise the same expected number of different labeling atom masses,then any pixel comprising more than the expected number of targetbarcode labeling atom masses may be disregarded (i.e., it may not bepossible to deconvolve which combination of target barcodes were presentat that spot). In certain aspects, some mass tags may only comprise asingle labeling atom mass selected from a first subset of masses, andother mass tags may be target barcoded and may comprise a uniquecombination of labeling atom masses selected from a second subset ofmasses that does not overlap with the first subset of masses. Forexample, mass tags to targets at high abundance may not be targetbarcoded. However, by controlling the concentration of mass tags and/orcompeting with affinity reagents that are not mass tagged, the instanceof target barcoded mass tags that bind high abundance targets could bereduced to a level that allows for single copy detection per pixel. Insome cases, a first imaging of the sample at a lower resolution and/orlower density of sampling may allow for non target barcoded mass tags tobe detected, and for the identification of regions of interest. A higherresolution and/or higher density sampling may then be performed at theidentified regions of interest to detect single copies of targetbarcoded mass tags.

To detect many targets at single molecule resolution in single cells,many pixels may need to be sampled from the cell. For example, a 10micron cell that is roughly spherical could be sampled close to 1billion times with a 10 nm spot size, provided the same location of thecell (e.g., X-Y coordinate) is sampled multiple times (e.g., atdifferent Z-depths). A subset of pixels would have single targetbarcoded mass tags, and of those, many may have recurring targetbarcoded mass tags (which could be counted to quantify the expressionlevel of the target). For example, if 1 million pixels are detected in acell, and 10% of those pixels have a single copy of a target barcodemass tag, and the average number of each target barcode mass tag in thecell is 100, then there 1000 different target barcode mass tags could bedetected in the cell.

Mass tags described above may be provided in a kit, optionally alongsideor bound to affinity reagents and/or oligonucleotides so as to bindtargets in a sample.

Definitions

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The term “about” in relation to a numerical value x is optional andmeans, for example, x+10%.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the disclosure.

EXAMPLES Example 1

Protocol

The mouse gut was immersion fixed following organ harvesting using 2%PFA and 2.5% glutaraldehyde. The tissue was post-fixed with 2% OsmiumTetroxide for 1 hour. After dehydration the tissue sample was embeddedin EPON resin and sectioned.

The 1000 nm tissue section was subjected to laser ablation and analyzedfor the presence of osmium isotopes by Hyperion imaging system.

Results

FIG. 11 illustrates detection of abundant osmium isotopes (¹⁸⁸Os, ¹⁸⁹Os,¹⁹⁰Os and ¹⁹²Os), as well as isotopes that are naturally low abundant(0.02% ¹⁸⁴Os, 1.59% ¹⁸⁶Os, 1.96% ¹⁸⁷Os) present in the mouse gut tissueprocessed according to the standard immunoelectron microscopy protocols.The gut morphology is well defined with a dense outer layer of smoothmuscle (upper left corner), mucosal epithelium with goblet cells andscattered stroma and immune cells beneath the epithelium.

Thus, electron microscopy 1 μm sections treated with 2% osmium tetroxidecan be imaged for structural detail. The osmium tetroxide concentrationused to fix tissue is determined not to be oversaturating for the IMCdetector.

IMC settings: Mouse gut resin, He=1050, Ar=0_9, Z=2871, atten=30, 200um×200 um, 200 Hz 200 pulses 200 um-sec, area1

A larger area of the mouse gut was also ablated. The image of FIG. 12shows 2100×1087 um area, at 250 nm step size.

The invention claimed is:
 1. A method of analyzing a sample by imagingmass spectrometry, comprising: radiating the sample in a sample chamberwith a first energy source to release material from the sample; allowingthe material to expand past the point of significant chargeneutralization once ionized; ionizing the expanded material with anionizing laser to: form a microplasma near the sample surface, andgenerate elemental ions; and analyzing the elemental ions by massspectrometry.
 2. The method of claim 1, wherein allowing the material toexpand comprises allowing the material to expand into a partial vacuumin the sample chamber.
 3. The method of claim 2, wherein the partialvacuum is 10-10,000 Pa.
 4. The method of claim 1, wherein allowing thematerial to expand comprises allowing the material to expand for 100 psto 10 ns before ionizing.
 5. The method of claim 1, wherein the ionizinglaser is an IR laser or a visible laser.
 6. The method of claim 1,wherein the ionizing laser is a picosecond laser or a femtosecond laser.7. The method of claim 1, further comprising directing ions resultingfrom the microplasma using ion optics positioned near the sample.
 8. Themethod of claim 1, wherein analyzing is by a time-of-flight massspectrometer or a magnetic sector mass spectrometer.
 9. The method ofclaim 1, further comprising analyzing the sample by imaging masscytometry.
 10. The method of claim 1, wherein the first energy source isa laser.
 11. The method of claim 10, wherein the laser has a wavelengthof less than 300 nm.
 12. The method of claim 10, wherein the laser isfocused with an objective lens of numerical aperture of at least 0.9.13. The method of claim 10, wherein the laser is focused to a spot sizeof 250 nm or less.
 14. The method of claim 10, wherein the laser ispositioned on the opposite side of a sample stage from the sample. 15.The method of claim 1, wherein the first energy source is an ion beam.16. The method of claim 1, wherein the first energy source is anelectron beam.
 17. The method of claim 1, further comprising ablatingthe material from the sample using the first energy source, wherein thefirst energy source comprises a first laser source and a second lasersource.
 18. The method of claim 1, wherein the sample is a biologicalsample.
 19. The method of claim 18, wherein the biological sample is atissue sample that has a thickness of 100 nm or below.
 20. The method ofclaim 18, wherein the biological sample comprises labelling atoms.